Human antibody molecules for il-13

ABSTRACT

Specific binding members, in particular human anti-IL-13 antibody molecules and especially those which neutralize IL-13 activity. Methods for using anti-IL-13 antibody molecules in diagnosis or treatment of IL-13 related disorders, including asthma, atopic dermatitis, allergic rhinitis, fibrosis, inflammatory bowel disease and Hodgkin&#39;s lymphoma.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This is a divisional of U.S. patent application Ser. No. 15/058,612,filed on Mar. 2, 2016, which is a divisional of U.S. patent applicationSer. No. 12/941,677, filed on Nov. 8, 2010, now U.S. Pat. No. 9,315,575,which is a divisional of U.S. patent application Ser. No. 10/564,647,filed on Jul. 19, 2006, now U.S. Pat. No. 7,829,090, which is a U.S.national stage entry of International Patent Application No.PCT/GB2004/003059, filed on Jul. 15, 2004, which claims priority to U.S.Provisional Patent Application No. 60/573,791, filed on May 24, 2004,U.S. Provisional Patent Application No. 60/558,216, filed on Mar. 31,2004, U.S. Provisional Patent Application No. 60/487,512, filed on Jul.15, 2003, and GB Patent Application No. 0407315.1, filed on Mar. 31,2004, the entire contents of all of which are fully incorporated hereinby reference.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 95,782 Byte ASCII (Text) file named“2017_11_27_208689-9017-US06-SEQ-LIST.txt”, created on Nov. 27, 2017.

The present invention relates to specific binding members, in particularhuman anti-IL-13 antibody molecules and especially those whichneutralise IL-13 activity. It further relates to methods for usinganti-IL-13 antibody molecules in diagnosis or treatment of IL-13 relateddisorders, including asthma, atopic dermatitis, allergic rhinitis,fibrosis, inflammatory bowel disease and Hodgkin's lymphoma.

Preferred embodiments of the present invention employ the antibody VHand/or VL domain of the antibody molecule herein termed BAK502G9 andother antibody molecules of the BAK502G9 lineage and of the BAK278D6lineage, as herein defined. Further preferred embodiments employcomplementarity determining regions (CDRs) of the BAK278D6 lineage, andpreferably BAK502G9, especially VH CDR3 in other antibody frameworkregions. Further aspects of the present invention provide forcompositions containing specific binding members of the invention, andtheir use in methods of inhibiting or neutralising IL-13, includingmethods of treatment of the human or animal body by therapy.

The present invention provides antibody molecules of particular value inbinding and neutralising IL-13, and thus of use in any of a variety oftherapeutic treatments, as indicated by the experimentation containedherein and further by the supporting technical literature.

Interleukin (IL)-13 is a 114 amino acid cytokine with an unmodifiedmolecular mass of approximately 12 kDa [1,2]. IL-13 is most closelyrelated to IL-4 with which it shares 30% sequence similarity at theamino acid level. The human IL-13 gene is located on chromosome 5q31adjacent to the IL-4 gene [1][2]. This region of chromosome 5q containsgene sequences for other Th2 lymphocyte derived cytokines includingGM-CSF and IL-5, whose levels together with IL-4 have been shown tocorrelate with disease severity in asthmatics and rodent models ofallergic inflammation [3][4][5][6][7][8].

Although initially identified as a Th2 CD4+ lymphocyte derived cytokine,IL-13 is also produced by Th1 CD4+ T-cells, CD8+ T lymphocytes NK cells,and non-T-cell populations such as mast cells, basophils, eosinophils,macrophages, monocytes and airway smooth muscle cells.

IL-13 is reported to mediate its effects through a receptor system thatincludes the IL-4 receptor α chain (IL-4Rα), which itself can bind IL-4but not IL-13, and at least two other cell surface proteins, IL-13Rα1and IL-13Rα2 [9][10]. IL-13Rα1 can bind IL-13 with low affinity,subsequently recruiting IL-4Rα to form a high affinity functionalreceptor that signals [11][12]. The Genbank database lists the aminoacid sequence and the nucleic acid sequence of IL-13Rα1 as NP 001551 andY10659 respectively. Studies in STAT6 (signal transducer and activatorof transcription 6) -deficient mice have revealed that IL-13, in amanner similar to IL-4, signals by utilising the JAK-STAT6 pathway[13][14]. IL-13Rα2 shares 37% sequence identity with IL-13Rα1 at theamino acid level and binds IL-13 with high affinity [15][16]. However,IL-13Rα2 has a shorter cytoplasmic tail that lacks known signallingmotifs. Cells expressing IL-13Rα2 are not responsive to IL-13 even inthe presence of IL-4Rα [17]. It is postulated, therefore, that IL-13Rα2acts as a decoy receptor regulating IL-13 but not IL-4 function. This issupported by studies in IL-13Rα2 deficient mice whose phenotype wasconsistent with increased responsiveness to IL-13 [18][19]. The Genbankdatabase lists the amino acid sequence and the nucleic acid sequence ofIL-13Rα2 as NP 000631 and Y08768 respectively.

The signalling IL-13Rα1/IL-4Rα receptor complex is expressed on humanB-cells, mast cells, monocyte/macrophages, dendritic cells, eosinophils,basophils, fibroblasts, endothelial cells, airway epithelial cells andairway smooth muscle cells.

Bronchial asthma is a common persistent inflammatory disease of the lungcharacterised by airways hyper-responsiveness, mucus overproduction,fibrosis and raised serum IgE levels. Airways hyper-responsiveness (AHR)is the exaggerated constriction of the airways to non-specific stimulisuch as cold air. Both AHR and mucus overproduction are thought to beresponsible for the variable airway obstruction that leads to theshortness of breath characteristic of asthma attacks (exacerbations) andwhich is responsible for the mortality associated with this disease(around 2000 deaths/year in the United Kingdom).

The incidence of asthma, along with other allergic diseases, hasincreased significantly in recent years [20][21]. For example,currently, around 10% of the population of the United Kingdom (UK) hasbeen diagnosed as asthmatic.

Current British Thoracic Society (BTS)and Global Initiative for Asthma(GINA) guidelines suggest a stepwise approach to the treatment of asthma[22, 23]. Mild to moderate asthma can usually be controlled by the useof inhaled corticosteroids, in combination with beta-agonists orleukotriene inhibitors. However, due to the documented side effects ofcorticosteroids, patients tend not to comply with the treatment regimewhich reduces the effectiveness of treatment [24-26].

There is a clear need for new treatments for subjects with more severedisease, who often gain very limited benefit from either higher doses ofinhaled or oral corticosteroids recommended by asthma guidelines. Longterm treatment with oral corticosteroids is associated with side effectssuch as osteoporosis, slowed growth rates in children, diabetes and oralcandidiasis [88]. As both beneficial and adverse effects ofcorticosteroids are mediated via the same receptor, treatment is abalance between safety and efficacy. Hospitalisation of these patients,who represent around 6% of the UK asthma population, as a result ofsevere exacerbations accounts for the majority of the significanteconomic burden of asthma on healthcare authorities [89].

It is believed that the pathology of asthma is caused by ongoing Th2lymphocyte mediated inflammation that results from inappropriateresponses of the immune system to harmless antigens. Evidence has beenaccrued which implicates IL-13, rather than the classical Th2 derivedcytokine IL-4, as the key mediator in the pathogenesis of establishedairway disease.

Administration of recombinant IL-13 to the airways of naivenon-sensitised rodents caused many aspects of the asthma phenotypeincluding airway inflammation, mucus production and AHR[27][28][29][30]. A similar phenotype was observed in a transgenic mousein which IL-13 was specifically overexpressed in the lung. In this modelmore chronic exposure to IL-13 also resulted in fibrosis [31].

Further, in rodent models of allergic disease many aspects of the asthmaphenotype have been associated with IL-13. Soluble murine IL-13Rα2, apotent IL-13 neutraliser, has been shown to inhibit AHR, mucushypersecretion and the influx of inflammatory cells which arecharacteristics of this rodent model [27][28][30]. In complementarystudies, mice in which the IL-13 gene had been deleted, failed todevelop allergen-induced AHR. AHR could be restored in these IL-13deficient mice by the administration of recombinant IL-13. In contrast,IL-4 deficient mice developed airway disease in this model [32][33].

Using a longer-term allergen-induced pulmonary inflammation model, Taubeat al. demonstrated the efficacy of soluble murine IL-13Rα2 againstestablished airway disease [34]. Soluble murine IL-13Rα2 inhibited AHR,mucus overproduction and to a lesser extent airway inflammation. Incontrast, soluble IL-4Rα, which binds and antagonises IL-4, had littleeffect on AHR or airway inflammation in this system [35]. These findingswere supported by Blease et al. who developed a chronic fungal model ofasthma in which polyclonal antibodies against IL-13 but not IL-4 wereable to reduce mucus overproduction, AHR and subepithelial fibrosis[36].

A number of genetic polymorphisms in the IL-13 gene have also beenlinked to allergic disease. In particular, a variant of the IL-13 genein which the arginine residue at amino acid 130 is substituted withglutamine (R130Q) has been associated with bronchial asthma, atopicdermatitis and raised serum IgE levels [37] [38] [39] [40]. Thisparticular IL-13 variant is also referred to as the R110Q variant(arginine residue at amino acid 110 is substituted with glutamine) bysome groups who exclude the 20 amino acid signal sequence from the aminoacid count. Arima et al, [41] report that this variant is associatedwith raised levels of IL-13 in serum. The IL-13 variant (R130Q) andantibodies to this variant are discussed in WO 01/62933. An IL-13promoter polymorphism, which alters IL-13 production, has also beenassociated with allergic asthma [42].

Raised levels of IL-13 have also been measured in human subjects withasthma, atopic rhinitis (hay fever), allergic dermatitis (eczema) andchronic sinusitis. For example levels of IL-13 were found to be higherin bronchial biopsies, sputum and broncho-alveolar lavage (BAL) cellsfrom asthmatics compared to control subjects [43][44][45][46]. Further,levels of IL-13 in BAL samples increased in asthmatic individuals uponchallenge with allergen [47][48]. The IL-13 production capacity ofCD4(+) T cells has further been shown to be useful marker of risk forsubsequent development of allergic disease in newborns [49].

Li et al [114] have recently reported affects of a neutralisinganti-mouse IL-13 antibody in a chronic mouse model of asthma. Chronicasthma-like response (such as AHR, severe airway inflammation, hypermucus productions) was induced in OVA sensitised mice. Li et al reportthat administration of an IL-13 antibody at the time of each OVAchallenge suppresses AHR, eosinophil infiltration, serum IgE levels,proinflammatory cytokine/chemokine levels and airway remodelling [14].

In summary, these data provide indication that IL-13 rather than IL-4 isa more attractive target for the treatment of human allergic disease.

IL-13 may play a role in the pathogenesis of inflammatory bowel disease.Heller et al.[116] report that neutralisation of IL-13 by administrationof soluble IL-13Rα2 ameliorated colonic inflammation in a murine modelof human ulcerative colitis [116]. Correspondingly, IL-13 expression washigher in rectal biopsy specimens from ulcerative colitis patients whencompared to controls [117].

Aside from asthma, IL-13 has been associated with other fibroticconditions. Increased levels of IL-13, up to a 1000 fold higher thanIL-4, have been measured in the serum of patients with systemicsclerosis [50] and in BAL samples from patients affected with otherforms of pulmonary fibrosis [51]. Correspondingly, overexpression ofIL-13 but not IL-4 in the mouse lung resulted in pronounced fibrosis[52][53]. The contribution of IL-13 to fibrosis in tissues other thanthe lung has been extensively studied in a mouse model ofparasite-induced liver fibrosis. Specific inhibition of IL-13 byadministration of soluble IL-13Rα2 or IL-13 gene disruption, but notablation of IL-4 production prevented fibrogenesis in the liver[54][55][56].

Chronic Obstructive Pulmonary Disease (COPD) includes patientpopulations with varying degrees of chronic bronchitis, small airwaydisease and emphysema and is characterised by progressive irreversiblelung function decline that responds poorly to current asthma basedtherapy [90]. The incidence of COPD has risen dramatically in recentyears to become the fourth leading cause of death worldwide (WorldHealth Organisation). COPD therefore represents a large unmet medicalneed.

The underlying causes of COPD remain poorly understood. The “Dutchhypothesis” proposes that there is a common susceptibility to COPD andasthma and therefore, that similar mechanisms may contribute to thepathogenesis of both disorders [57].

Zheng et al [58] have demonstrated that overexpression of IL-13 in themouse lung caused emphysema, elevated mucus production and inflammation,reflecting aspects of human COPD. Furthermore, AHR, an IL-13 dependentresponse in murine models of allergic inflammation, has been shown to bepredictive of lung function decline in smokers [59]. A link has alsobeen established between an IL-13 promoter polymorphism andsusceptibility to develop COPD [60].

The signs are therefore that IL-13 plays an important role in thepathogenesis of COPD, particularly in patients with asthma-like featuresincluding AHR and eosinophilia. mRNA levels of IL-13 have been shown tobe higher in autopsy tissue samples from subjects with a history of COPDwhen compared to lung samples from subjects with no reported lungdisease (J. Elias, Oral communication at American Thoracic SocietyAnnual Meeting 2002). In another study, raised levels of IL-13 weredemonstrated by immunohistochemistry in peripheral lung sections fromCOPD patients [91].

Hodgkin's disease is a common type of lymphoma, which accounts forapproximately 7,500 cases per year in the United States.

Hodgkin's disease is unusual among malignancies in that the neoplasticReed-Sternberg cell, often derived from B-cells, make up only a smallproportion of the clinically detectable mass. Hodgkin's disease-derivedcell lines and primary Reed-Sternberg cells frequently express IL-13 andits receptor [61]. As IL-13 promotes cell survival and proliferation innormal B-cells, it was proposed that IL-13 could act as a growth factorfor Reed-Sternberg cells. Skinnider et al. have demonstrated thatneutralising antibodies against IL-13 can inhibit the growth ofHodgkin's disease-derived cell lines in vitro [62]. This findingsuggested that Reed-Sternberg cells might enhance their own survival byan IL-13 autocrine and paracrine cytokine loop. Consistent with thishypothesis, raised levels of IL-13 have been detected in the serum ofsome Hodgkin's disease patients when compared to normal controls [63].IL-13 inhibitors may therefore prevent disease progression by inhibitingproliferation of malignant Reed-Sternberg cells.

Many human cancer cells express immunogenic tumour specific antigens.However, although many tumours spontaneously regress, a number evade theimmune system (immunosurveillance) by suppressing T-cell mediatedimmunity. Terabe et al.[64] have demonstrated a role of IL-13 inimmunosuppression in a mouse model in which tumours spontaneouslyregress after initial growth and then recur. Specific inhibition ofIL-13, with soluble IL-13Rα2, protected these mice from tumourrecurrence. Terabe et al [64] went on to show that IL-13 suppresses thedifferentiation of tumour specific CD8+ cytotoxic lymphocytes thatmediate anti-tumour immune responses.

IL-13 inhibitors may, therefore, be used therapeutically to preventtumour recurrence or metastasis. Inhibition of IL-13 has been shown toenhance anti-viral vaccines in animal models and may be beneficial inthe treatment of HIV and other infectious diseases [65].

It should be noted that generally herein reference to interleukin-13 orIL-13 is, except where context dictates otherwise, reference to humanIL-13. This is also referred to in places as “the antigen”. The presentinvention provides antibodies to human IL-13, especially humanantibodies, that are cross-reactive with non-human primate IL-13,including cynomolgus and rhesus monkey IL-13. Antibodies in accordancewith some embodiments of the present invention recognise a variant ofIL-13 in which the arginine residue at amino acid position 130 isreplaced by glutamine. In other aspects and embodiments the presentinvention provides specific binding members against murine IL-13,specifically mouse IL-13.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows neutralisation potency (% inhibition) of BAK167A11 (closedsquares) and its derivative BAK615E3 (open squares) as scFv against 25ng/ml human IL-13 in the TF-1 cell proliferation assay. The trianglesrepresent an irrelevant scFv. Data represent the mean with standarderror bars of triplicate determinations within the same experiment.

FIG. 2 shows the neutralisation potency (% inhibition) of BAK278D6(closed squares) and its derivative BAK502G9 (open squares) as scFvagainst 25 ng/ml human IL-13 in the TF-1 cell proliferation assay. Thetriangles represent an irrelevant scFv. Data represent the mean withstandard error bars of triplicate determinations within the sameexperiment.

FIG. 3 shows the neutralisation potency (% inhibition) of BAK209B11(closed squares) as a scFv against 25 ng/ml murine IL-13 in the TF-1cell proliferation assay. The triangles represent an irrelevant scFv.Data represent the mean with standard error bars of triplicatedeterminations within the same experiment.

FIG. 4 shows the neutralisation potency (% inhibition) of BAK278D6(closed squares) as a scFv against IL-13 in the TF-1 cell proliferationassay. The triangles represent an irrelevant scFv. Data represent themean with standard error bars of triplicate determinations within thesame experiment.

FIG. 4A show potency against 25 ng/ml human IL-13.

FIG. 4B shows potency against 25 ng/ml human variant IL-13.

FIG. 4C shows potency against 50 ng/ml non-human primate IL-13.

FIG. 5 shows a comparison of the potency of anti-human IL-13 antibodiesin the TF-1 proliferation assay. Data represent the mean neutralisationpotency with standard error bars over 5-7 experiments against 25 ng/mlhuman IL-13. The performance relative to the commercially availableantibody, B-B13, was evaluated statistically by performing a one-wayANOVA with Dunnett's test. *P<0.05, **P<0.01 compared to B-B13.

FIG. 6 shows the neutralisation potency (% inhibition) of BAK502G9(closed squares), BAK1167F2 (closed triangles) and BAK1183H4 (closedinverted triangles) as human IgG4 against tagged IL-13 in the TF-1 cellproliferation assay. Open triangles represent an irrelevant IgG4. Datarepresent the mean with standard error bars of three separateexperiments.

FIG. 6A shows potency against 25 ng/ml human IL-13.

FIG. 6B shows potency against 25 ng/ml human variant IL-13.

FIG. 6C shows potency against 50 ng/ml non-human primate IL-13.

FIG. 7 shows the neutralisation potency (% inhibition) of BAK502G9(closed squares), BAK1167F2 (closed triangles), BAK1183H4 (closedinverted triangles) as human IgG4 and commercial anti-human IL-13antibodies (B-B13-open squares; JES10-5A2-open inverted triangles) inthe native IL-13 dependent HDLM-2 cell proliferation assay. Opentriangles represent an irrelevant IgG4. Data represent the mean withstandard error bars of triplicate determinations within the sameexperiment.

FIG. 8 shows a comparison of the potency of anti-human IL-13 antibodiesin the NHLF assay. Data represent the mean neutralisation potency (IC₅₀pM) with standard error bars over 4-5 experiments against 10 ng/ml humanIL-13 in the NHLF eotaxin release assay. The performance relative to thecommercially available antibody, B-B13, was evaluated statistically byperforming a one-way ANOVA with Dunnett's test. *P<0.05, **P<0.01compared to B-B13.

FIG. 9 shows the neutralisation potency (% inhibition) of BAK502G9(closed squares), BAK1167F2 (closed triangles), BAK1183H4 (closedinverted triangles) as human IgG4 against VCAM-1 upregulation on thesurface of HUVEC in response to 10 ng/ml human IL-13. Open trianglesrepresent irrelevant IgG4. Data represent the mean with standard errorbars of triplicate determinations within the same experiment.

FIG. 10 shows the neutralisation potency (% inhibition) of BAK502G9(closed squares), BAK1167F2 (closed triangles), BAK1183H4 (closedinverted triangles) as human IgG4 against eotaxin release from VCAM-1upregulation on the surface of HUVEC in response to either Ing/ml humanIL-4 (FIG. 10A) or 0.5 ng/ml human IL-1R (FIG. 10B). Open trianglesrepresent an irrelevant IgG4. Data represent the mean with standarderror bars of triplicate determinations within the same experiment.

FIG. 11 shows the neutralisation potency (% inhibition) of BAK209B11(squares) as a human IgG4 against 1 ng/ml murine IL-13 in the factordependent B9 cell proliferation assay. Open triangles represent anirrelevant IgG4. Data represent the mean with standard error bars oftriplicate determinations within the same experiment.

FIG. 12 shows the relative level of IL-13 in lung homogenates fromsensitised (s) (right-hand bar) and non-sensitised (ns) (left-hand bar)mice post challenge in a murine model of acute pulmonary allergicinflammation. The effect of sensitisation was statistically evaluated byperforming Student's t-test using quantity of IL-13 data. *<0.05.**<0.01 compared to non-sensitised control animals (n=5-6 mice). Datarepresent the mean with standard error bars.

FIG. 13 illustrates the effects of i.v. administration of

BAK209B11 as human IgG4 in different amounts compared to an isotypematched IgG4 irrelevant control antibody on ovalbumin induced leukocyterecruitment to the lung in ovalbumin sensitised mice. The number ofleukocytes is shown (×10⁴). The effect of antibody treatment wasstatistically evaluated by performing one way ANOVA with Dunnett's testusing differential cell count data. *<0.05. **<0.01 compared toovalbumin challenged PBS control animals (=0% inhibition; n=5-8 mice).Data represent the mean with standard error bars.

FIG. 14 illustrates the effects of i.v. administration of BAK209B11 ashuman IgG4 in different amounts compared to an isotype matched IgG4irrelevant control antibody on ovalbumin induced eosinophil recruitmentto the lung in ovalbumin sensitised mice. The number of eosinophils isshown (×10⁴). The effect of antibody treatment was statisticallyevaluated by performing one way ANOVA with Dunnett's test usingdifferential cell count data. *<0.05. **<0.01 compared to ovalbuminchallenged PBS control animals (=0% inhibition; n=5-8 mice). Datarepresent the mean with standard error bars.

FIG. 15 illustrates the effects of i.v. administration of BAK209B11 ashuman IgG4 in different amounts compared to an isotype matched IgG4irrelevant control antibody on ovalbumin induced neutrophil recruitmentto the lung in ovalbumin sensitised mice. The number of neutrophils isshown (×10⁴). The effect of antibody treatment was statisticallyevaluated by performing one way ANOVA with Dunnett's test usingdifferential cell count data. *<0.05. **<0.01 compared to ovalbuminchallenged PBS control animals (=0% inhibition; n=5-8 mice). Datarepresent the mean with standard error bars.

FIG. 16 illustrates the effects of i.v. administration of BAK209B11 ashuman IgG4 in different amounts compared to an isotype matched IgG4irrelevant control antibody on ovalbumin induced lymphocyte recruitmentto the lung in ovalbumin sensitised mice. The induction of lymphocyteswas dose dependently inhibited by BAK209B11 with maximal inhibition at 3μg/ml of BAK209B11. The effect of antibody treatment was statisticallyevaluated by performing one way ANOVA with Dunnett's test usingdifferential cell count data. *<0.05. **<0.01 compared to ovalbuminchallenged PBS control animals (=0% inhibition; n=5-8 mice). Datarepresent the mean with standard error bars.

FIG. 17 illustrates the effects of i.v. administration of BAK209B11 ashuman IgG4 in different amounts compared to an isotype matched IgG4irrelevant control antibody on ovalbumin induced monocyte/macrophagerecruitment to the lung in ovalbumin sensitised mice. There was nosignificant increase in the levels of monocytes/macrophages ofsensitised animals when compared with control animals. However, suchbackground levels of these cells were depressed by 36 μg/ml BAK209B11 insensitised animals. The effect of antibody treatment was statisticallyevaluated by performing one way ANOVA with Dunnett's test usingdifferential cell count data. *<0.05. **<0.01 compared to ovalbuminchallenged PBS control animals (=0% inhibition; n=5-8 mice). Datarepresent the mean with standard error bars.

FIG. 18 shows the effects of a commercial anti-IL-13 neutralisingantibody JES10-5A2 on the influx of cells (number of leukocytes is shown(×10⁴)) into the murine airpouch elicited by administration ofbacterially derived recombinant human IL-13. The effect of antibodytreatment was statistically evaluated by performing one way ANOVA withDunnett's test using differential cell count data. *<0.05. **<0.01compared to CMC control animals (=0% inhibition; n=11-13 mice). Datarepresent the mean with standard error bars.

FIG. 19 shows an sequence alignment of cynomolgus IL-13 against humanIL-13 amino acid sequences. The seven amino acid residues that differbetween human and cynomolgus IL-13 are shaded. Rhesus and cynomolgusIL-13 have an identical amino acid sequence.

FIG. 20 illustrates the effects of single 10 mg/kg i.v bolus dose ofBAK502G9 as human IgG4 on serum IgE levels in 4 allergic butnon-challenged cynomolgus primates (2 male/2 female) over 29 days. SerumIgE concentration is significantly reduced from 100% (predose) to 66±10%of control values (p<0.05), at 4 and 5 days after dosing. This loweringof serum IgE concentration recovers to 88±8% of control levels by day22. *=p<0.05 as compared to predose IgE levels, repeated measures ANOVAfollowed by Dunnett's multiple comparisons test (n=4 animals).

FIG. 20B shows relative serum IgE levels of male and female cynomolgusprimates versus time following a single 10 mg/kg intravenous dose ofBAK502G9. Relative serum IgE data are expressed as arithmetic mean±SEMpercentage of baseline value.

FIG. 21 illustrates the effects of intraperitoneal administration ofBAK209B11 in different amounts (H=237 μg/day, M=23.7 μg/day and L=2.37μg/day) compared with an isotype matched IgG1 irrelevant controlantibody on the lung function of ovalbumin sensitised and challengedmice. In FIG. 21A lung function is represented by log PC₅₀s (logmethacholine concentration required to increase baseline PenH by 50%)before any treatment (day 0) and post sensitisation, challenge and drugtreatment (day 25). FIG. 21A shows the raw data used to calculate thestudy endpoint, shown in FIG. 21B (Delta log PC₅₀). Data represent themean with standard error bars of n=8.

In FIG. 21B changing lung function is shown by a change in an individualmouse's log PC₅₀ (delta log PC₅₀). Delta log PC₅₀ is defined as anindividuals change in log PC₅₀ at day 25 verus day 0. Data representgroup mean delta log PC₅₀ (individual changes averaged within treatmentgroups) with standard error bars. The effect of antibody treatment wasstatistically evaluated by performing one way ANOVA with Dunnett's testusing delta log PC₅₀ data. **p<0.01 compared to ovalbumin sensitised andchallenged control animals (n=8 mice).

FIG. 22 illustrates the effects of local (i.po.) and systemic (i.v.)administration of BAK502G9 as human IgG4 in different amounts comparedto an isotype matched IgG4 irrelevant control antibody on the totalleukocyte recruitment (FIG. 22A) and eosinophil recruitment (FIG. 22B)into the air pouch of BALB/C mice. Data represent the mean with standarderror bars of n=10. The effect of antibody treatment was statisticallyevaluated by performing one way ANOVA with Dunnett's test usinglog-transformed data. *p<0.05, **p<0.01 compared to huIL-13 challengedmice (n=10).

FIG. 23: illustrates the effects of i.p. administration of BAK502G9 ashuman IgG4 compared to an isotype matched IgG4 irrelevant controlantibody on the development of AHR following intratrachealadministration of human IL-13 to the airways of mice. The effect ofantibody treatment was statistically evaluated by performing one wayANOVA with Dunnett's test using PC₂₀₀ Methacholine data. *<0.05. **<0.01compared to the human IL-13 positive control group (n=6-8 mice). Datarepresent the mean with standard error bars.

FIG. 24 shows the neutralisation potency (% maximal response) ofBAK502G9 (closed squares) as IgG4 against 30 ng/ml IL-13 in a human Bcell IgE production assay. Open squares represent an irrelevant IgG4.Data represent the mean with standard error bars of six donors fromseparate experiments.

FIG. 25 shows the effects of BAK502G9 on IL-13 induced potentiation ofagonist induced Ca²⁺ signalling in bronchial smooth muscle cells. Thearea under the curve (AUC) of the Ca²⁺ signalling response to histaminewas determined for each antibody +/−IL-13 pre-treatment condition.Combined data from three independent experiments are shown forirrelevant antibody CAT-001 (a) and BAK502G9 (b) as the percentagedifference versus untreated cells of AUC±SD (ns=not significant(p>0.05), *p<0.05, **p<0.01). The results were statistically evaluatedutilising a one-way analysis of variance (ANOVA) with Bonferroni'smultiple comparisons post-test.

FIG. 26 shows effects of phase II administered BAK502G9.

FIG. 26A shows effect on AHR as measured by change in area under thehistamine dose response curve (n=14).

FIG. 26B shows effect on AHR as measured by change in PC₃₀ (n=18).

FIG. 26C shows effect on antigen priming (n=20).

FIG. 26D shows effect on BAL inflammation (n=21).

FIG. 27 shows effect of BAK502G9 on IL-13-induced CD23 expression. Dataare presented as a percentage of the response to IL-13 alone (100%) andexpressed as mean±SEM % control of 6 separate experiments from 6individual donors (performed in triplicate).

FIG. 28 shows effect of BAK502G9 and irrelevant IgG4 on IL-13 and/orIL-4 induced PBMC CD23 expression. Data are presented as a percentage ofthe response to IL-4 alone (100%) and expressed as mean±SEM % control of4 separate experiments from 4 individual donors (performed intriplicate).

FIG. 29A shows effect of BAK502G9 on NHLF eotaxin-1 production inducedby 48 h culture with IL-13/TNF-α/TGF-β1 containing media. Data are shownas an arithmetic mean±SEM from triplicate determinations of the mediaused in this study to induce leukocyte shape change.

FIG. 29B shows effect of BAK502G9 on shape change of human eosinophilsinduced by 1:16 diultion of conditioned media. Data points representedare mean±SEM %blank media shape change from separate experiments fromfour individual donors.

FIG. 30 shows alignment of human IL-13 against murine IL13 highlightingthe mutations that were introduced into human IL-13 to produce the firstpanel of IL-13 chimaeras. The four alpha helices are highlighted inboxes and loop 1 and loop 3 are labelled. Five chimeric proteins wereproduced where helices B, C and D, and loops 1 and loop 3 were replacedwith the murine sequence. Four further chimeric proteins were producedand numbered according to the amino acid in the human pre-protein (notto the numbering of the multiple aligment above) where arginine atresidue 30 (position 34 above) was mutated, residues 33 and 34 (position37 and 38 above) were mutated, residues 37 and 38 (VH) were mutated(position 41 and 42 above), and residues 40 and 41 (TQ) were mutated(position 44 and 45 above).

FIG. 31 shows alignment of human IL-13 against murine IL-13 highlightingthe mutations that were introduced into human IL-13 to produce thesecond panel of IL-13 chimaeras. Six chimaeras were produced where thehuman residue(s) were substituted for the murine residue(s) (highlightedwith boxes). Four further chimeric proteins were produced (numbering isaccording to the amino acid position in the human pre-protein) whereleucine at residue 58 (62 in above figure) was mutated, leucine atresidue 119 (residue 123 above) was mutated, lysine at position 123(residue 127 above) was mutated, and arginine at residue 127 (residue132 above was mutated.

FIG. 32 shows mutations made to human IL-13. Mutations in dark greyreduced binding to BAK502G9, mutations in light grey did not alterbinding. Linear sequence of pre-human IL-13 with the mutated residuesindicated.

In various aspects and embodiments of the invention there is providedthe subject-matter of the claims included below.

The present invention provides specific binding members for IL-13, inparticular human and/or primate IL-13 and/or variant IL-13 (Q130R), andmurine IL-13. Preferred embodiments within the present invention areantibody molecules, whether whole antibody (e.g. IgG, such as IgG4) orantibody fragments (e.g. scFv, Fab, dAb). Antibody antigen bindingregions are provided, as are antibody VH and VL domains. Within VH andVL domains are provided complementarity determining regions,

CDR's, which may be provided within different framework regions, FR's,to form VH or VL domains as the case may be. An antigen binding site mayconsist of an antibody VH domain and/or a VL domain.

An antigen binding site may be provided by means of arrangement of CDR'son non-antibody protein scaffolds such as fibronectin or cytochrome Betc. [115, 116]. Scaffolds for engineering novel binding sites inproteins have been reviewed in detail by Nygren et al [116]. Proteinscaffolds for antibody mimics are disclosed in WO/0034784 in which theinventors describe proteins (antibody mimics) which include afibronectin type III domain having at least one randomised loop. Asuitable scaffold into which to graft one or more CDR's, e.g. a set ofHCDR's, may be provided by any domain member of the immunoglobulin genesuperfamily.

Preferred embodiments of the present invention are in what is termedherein the “BAK278D6 lineage”. This is defined with reference to a setof six CDR sequences of BAK278D6 as follows: HCDR1 (SEQ ID NO: 1), HCDR2(SEQ ID NO: 2), HCDR3 (SEQ ID NO: 3), LCDR1 (SEQ ID NO: 4), LCDR2 (SEQID NO: 5) and LCDR3 (SEQ ID NO: 6). In one aspect, the present inventionprovides a specific binding member for human IL-13, comprising anantibody antigen-binding site which is composed of a human antibody VHdomain and a human antibody VL domain and which comprises a set ofCDR's, wherein the VH domain comprises HCDR 1, HCDR2 and HCDR3 and theVL domain comprises LCDR1, LCDR2 and LCDR3, wherein the HCDR1 has theamino acid sequence of SEQ ID NO: 1, the HCDR2 has the amino acidsequence of SEQ ID NO: 2, the HCDR3 has the amino acid sequence of SEQID NO: 3, the LCDR1 has the amino acid sequence of SEQ ID NO: 4, theLCDR2 has the amino acid sequence of SEQ ID NO: 5, and the LCDR3 has theamino acid sequence of SEQ ID NO: 6; or wherein the set of CDR'scontains one or two amino acid substitutions compared with the set ofCDR's, wherein the HCDR1 has the amino acid sequence of SEQ ID NO: 1,the HCDR2 has the amino acid sequence of SEQ ID NO: 2, the HCDR3 has theamino acid sequence of SEQ ID NO: 3, the LCDR1 has the amino acidsequence of SEQ ID NO: 4, the LCDR2 has the amino acid sequence of SEQID NO: 5, and the LCDR3 has the amino acid sequence of SEQ ID NO: 6.

The set of CDR's wherein the HCDR1 has the amino acid sequence of SEQ IDNO: 1, the HCDR2 has the amino acid sequence of SEQ ID NO: 2, the HCDR3has the amino acid sequence of SEQ ID NO: 3, the LCDR1 has the aminoacid sequence of SEQ ID NO: 4, the LCDR2 has the amino acid sequence ofSEQ ID NO: 5, and the LCDR3 has the amino acid sequence of SEQ ID NO: 6,are herein referred to as the “BAK278D6 set of CDR's”. The HCDR1, HCDR2and HCDR3 within the BAK278D6 set of CDR's are referred to as the“BAK278D6 set of HCDR's” and the LCDR1, LCDR2 and LCDR3 within theBAK278D6 set of CDR's are referred to as the “BAK278D6 set of LCDR's”. Aset of CDR's with the BAK278D6 set of CDR's, BAK278D6 set of HCDR's orBAK278D6 LCDR's, or one or two substitutions therein, is said to be ofthe BAK278D6 lineage.

As noted, in one aspect the invention provides a specific binding memberfor human IL-13, comprising an antibody antigen-binding site which iscomposed of a human antibody VH domain and a human antibody VL domainand which comprises a set of CDR's, wherein the set of CDR's is theBAK278D6 set of CDR's or a set of CDR's containing one or twosubstitutions compared with the BAK278D6 set of CDR's.

In preferred embodiments, the one or two substitutions are at one or twoof the following residues within the CDRs of the VH and/or VL domains,using the standard numbering of Kabat [107].

31, 32, 34 in HCDR1

52, 52A, 53, 54, 56, 58, 60, 61, 62, 64, 65 in HCDR2

96, 97, 98, 99, 101 in HCDR3

26, 27, 28, 30, 31 in LCDR1

56 in LCDR2

95A, 97 in LCDR3

Preferred embodiments have two substitutions compared with the BAK278D6set of CDR's, at HCDR3 residue 99 and LCDR1 residue 27. Of theseembodiments, preferred embodiments have S substituted for N at HCDR3residue 99 and/or I substituted for N at LCDR 1 residue 27. Stillfurther embodiments have a substitution at HCDR3 residue 99 selectedfrom the group consisting of S, A, I, R, P and K, and/or a substitutionat LCDR1 residue 27 selected from the group consisting of I, L, M, C, V,K, Y, F, R, T, S, A, H and G.

In preferred embodiments one or two substitutions are made at one or twoof the following residues within the BAK278D6 set of CDR's in accordancewith the identified groups of possible substitute residues:

Position of Substitute Residue selected from the group substitutionconsisting of 31 in HCDR1: Q, D, L, G and E 32 in HCDR1: T 34 in HCDR1:V, I and F 52 in HCDR2: D, N, A, R, G and E 52A in HCDR2: D, G, T, P, Nand Y 53 in HCDR2: D, L, A, P, T, S, I and R 54 in HCDR2: S, T, D, G, Kand I 56 in HCDR2: T, E, Q, L, Y, N, V, A, M and G 58 in HCDR2: I, L, Q,S, M, H, D and K 60 in HCDR2: R 61 in HCDR2: R 62 in HCDR2: K and G 64in HCDR2: R 65 in HCDR2: K 96 in HCDR3: R and D 97 in HCDR3: N, D, T andP 98 in HCDR3: R 99 in HCDR3: S, A, I, R, P and K 101 in HCDR3: Y 26 inLCDR1: D and S 27 in LCDR1: I, L, M, C, V, K, Y, F, R, T, S, A, H and G28 in LCDR1: V 30 in LCDR1: G 31 in LCDR1: R 56 in LCDR2: T 95A inLCDR3: N 97 in LCDR3: I

Preferred embodiments have the BAK278D6 set of CDR's with a substitutionof S for N at residue 99 within HCDR3 and I for N at residue 27 withinLCDR 1. The set of CDR's thus defined is as follows: HCDR1-SEQ ID NO: 7;HCDR2-SEQ ID NO: 8, HCDR3-SEQ ID NO: 9; LCDR1-SEQ ID NO: 10, LCDR2-SEQID NO: 11; LCDR3-SEQ ID NO: 12. This set of CDR's is herein referred toas the “BAK502G9 set of CDR's”.

Further preferred embodiments have the BAK278D6 set of CDR's with one ortwo substitutions within the CDR's, with the proviso that the pair ofsubstitutions of S for N at residue 99 within HCDR3 and I for N atresidue 27 within LCDR 1 is excluded.

Other preferred embodiments are as follows: BAK 1166G2: HCDR1-SEQ ID NO:67, HCDR2-SEQ ID NO: 68, HCDR3-SEQ ID NO: 69, LCDR1-SEQ ID NO: 70,LCDR2-SEQ ID NO: 71; LCDR3-SEQ ID NO: 72.

BAK1167F2 HCDR1-SEQ ID NO: 61, HCDR2-SEQ ID NO:62, HCDR3-SEQ ID NO:63,LCDR1-SEQ ID NO: 64, LCDR2-SEQ ID NO: 65; LCDR3-SEQ ID NO: 66.

BAK1184C8: HCDR1-SEQ ID NO:73, HCDR2: SEQ ID NO:74, HCDR3-SEQ ID NO:75.LCDR1-SEQ ID NO: 76, LCDR2-SEQ ID NO: 77; LCDR3-SEQ ID NO: 78.

BAK1185E1: HCDR1-SEQ ID NO:79, HCDR2-SEQ ID NO:80, HCDR3-SEQ ID NO: 81.LCDR1-SEQ ID NO: 82, LCDR2-SEQ ID NO: 83; LCDR3-SEQ ID NO: 84.

BAK1167F4: HCDR1-SEQ ID NO: 85, HCDR2-SEQ ID NO:86, HCDR3-SEQ ID NO:87.LCDR1-SEQ ID NO: 88, LCDR2-SEQ ID NO: 89; LCDR3-SEQ ID NO: 90.

BAK1111D10: HCDR1-SEQ ID NO: 91, HCDR2-SEQ ID NO: 92, HCDR3-SEQ ID NO:93. LCDR1-SEQ ID NO: 94, LCDR2-SEQ ID NO: 95; LCDR3-SEQ ID NO: 96.

BAK1183H4: HCDR1-SEQ ID NO: 97, HCDR2-SEQ ID NO: 98, HCDR3-SEQ ID NO:99. LCDR1-SEQ ID NO: 100, LCDR2-SEQ ID NO: 101; LCDR3-SEQ ID NO: 102.

BAK1185F8: HCDR1-SEQ ID NO: 103, HCDR2-SEQ ID NO: 104, HCDR3-SEQ ID NO:105. LCDR1-SEQ ID NO: 106, LCDR2-SEQ ID NO: 107; LCDR3-SEQ ID NO: 108.All of these were derived from BAK502G9 by heavy chain CDR1 and CDR2randomisation and are thus of the BAK502G9 lineage.

A VH domain comprising a set of CDR's HCDR1, HCDR2 and HCDR3 of anyclone as shown in Table 1. Table 1 is also provided by the presentinvention, as is separately a VL domain comprising a set of CDR's LCDR1,LCDR2 and LCDR3 of the clones shown in Table 1. Preferably such a VHdomain is paired with such a VL domain, and most preferably the VH andVL domain pairings are the same as in the clones as set out in Table 1.

Further provided by the present invention is a VH domain comprising aset of CDR's HCDR1, HCDR2 and HCDR3 wherein the set of CDR's correspondsto that for any clone shown in Table 1 with one or two amino acidsubstitutions.

Further provided by the present invention is a VL domain comprising aset of CDR's LCDR1, LCDR2 and LCDR3 wherein the set of CDR's correspondsto that for any clone shown in Table 1 with one or two amino acidsubstitutions.

A specific binding member comprising an antibody antigen-binding domaincomprising such a VH and/or VL domain is also provided by the presentinvention.

The present inventors have identified the BAK278D6 lineage as providinghuman antibody antigen-binding domains against IL-13 which are ofparticular value. Within the lineage, BAK502G9 has been identified to beof special value. The BAK278D6 and BAK502G9 sets of CDR's have beenidentified already above.

Following the lead of computational chemistry in applying multivariatedata analysis techniques to the structure/property-activityrelationships [94], quantitative activity-property relationships ofantibodies can be derived using well-known mathematical techniques suchas statistical regression, pattern recognition and classification[95-100]. The properties of antibodies can be derived from empirical andtheoretical models (for example, analysis of likely contact residues orcalculated physicochemical property) of antibody sequence, functionaland three-dimensional structures and these properties can be consideredsingly and in combination.

An antibody antigen-binding site composed of a VH domain and a VL domainis formed by six loops of polypeptide: three from the light chainvariable domain (VL) and three from the heavy chain variable domain(VH). Analysis of antibodies of known atomic structure has elucidatedrelationships between the sequence and three-dimensional structure ofantibody combining sites[101,102]. These relationships imply that,except for the third region (loop) in VH domains, binding site loopshave one of a small number of main-chain conformations: canonicalstructures. The canonical structure formed in a particular loop has beenshown to be determined by its size and the presence of certain residuesat key sites in both the loop and in framework regions [101,102].

This study of sequence-structure relationship can be used for predictionof those residues in an antibody of known sequence, but of an unknownthree-dimensional structure, which are important in maintaining thethree-dimensional structure of its CDR loops and hence maintain bindingspecificity. These predictions can be backed up by comparison of thepredictions to the output from lead optimization experiments.

In a structural approach, a model can be created of the antibodymolecule [103] using an freely available or commercial package such asWAM [104]. A protein visualisation and analysis software package such asInsight II [105] or Deep View [106] may then be used to evaluatepossible substitutions at each position in the CDR. This information maythen be used to make substitutions likely to have a minimal orbeneficial effect on activity.

The present inventors analysed sequence data of the panel of clones forwhich the sets of CDR's are shown in Table 1.

The analysis tested the hypothesis that any binary combinations oflisted amino acid variations in the CDR's from the presented set of scFvvariants leads to a scFv variant with at least the starting potency ofthe parent scFv BAK278D6.

All scFv variants in the panel shown in Table 1 have been selected forimproved affinity and have been confirmed to display higher potency.

The observed amino acid variations can either be favourable,non-favourable or neutral in their effect on the starting potency ofscFv BAK278D6 in the TF-1 assay of 44 nM.

No linkage was observed between any two amino acid variations confirmingthat there was no synergy, either “positive” or “negative”, between anytwo selected amino acid variations.

There are four scenarios where such binary combination will fulfil thehypothesis and three scenarios where the hypothesis will not be valid.Synergistic amino acid variants are not considered as no linkage wasobserved.

The hypothesis is valid where:

A1: mutation 1 is favourable and mutation 2 is favourable

A2 : mutation 1 is favourable and mutation 2 is neutral

A3: mutation 1 is neutral and mutation 2 is neutral

A4: mutation 1 is favourable and mutation 2 is non-favourable (with theeffect of 1 outweighing the effect of 2) .

The hypothesis is not valid where:

B1: mutation 1 is non-favourable and mutation 2 is neutral

B2: mutation 1 is non-favourable and mutation 2 is non-favourable

B3: mutation 1 is favourable and mutation 2 is non-favourable (with theeffect of 2 outweighing the effect of 1).

For A4 to be possible, mutation 1 needs to be highly favourable tocounterbalance the negative effect of mutation 2 on potency. Since suchhighly favourable mutation would be present in the library of variantsused for selection, it would be selected for and would therefore appearfrequently in the panel of variants. Since synergy can be excluded, suchmutation would be beneficial in any kind of sequence context and shouldtherefore reappear in different scFv variants. An example for suchfrequent amino acid change is the change in the light chain CDR1Asn27Ile. However, this mutation on its own (in clone BAK531E2) has onlya modest 2-fold effect on potency (final IC50 of 23.2 nM). On its ownthis mutation would not allow the scenario depicted in A4, as it is nota highly favourable mutation. This suggests that every clone in thepresented set of IL-13 binding clones (Table 1) which has a light chainCDR1 Asn27Ile change along with one or more further mutations is atleast as potent as the variant having the single light chain CDR1Asn27Ile mutation. The other mutations are either neutral or positivebut do not have a negative or detrimental affect.

A further example is in the heavy chain CDR3 Asn99Ser (see Table 1). Asa clone carrying this particular single amino acid variation is notobserved, the potency of such a clone has been estimated to beapproximately 12.0 nM by the following rationale:

BAK278D6 potency is 44 nM. Alterations of VL CDR1 N27I+VH CDR3 N99S leadto BAK502G9 with potency 8 nM, i.e. 5.5 fold improvement.

BAK278D6 potency is 44 nM. Alteration of VL CDR1 N27I leads to BAK531E2with potency 23 nM, i.e. 1.9 fold improvement

BAK278D6 potency is 44 nM. Alteration VH CDR3 N99S to provide a possibleclone with potency 12.2 nM, i.e. 2.9 fold improvement (5.5/1.9=2.9).

The binary combination of heavy chain CDR3 Asn99Ser with light chainCDR1 Asn27Ile gives a scFv BAK0502G9 with a potency of 8 nM. As synergyis excluded, the contribution of heavy chain CDR3 Asn99Ser change inBAK502G9 is therefore additive.

Therefore every clone in the presented set of IL-13 binding clones(Table 1) which has a heavy chain CDR3 AsnH99Ser change along with oneor more further mutations would have a potency of at least 12 nM orgreater, within a permissive assay window of 2.5-fold for n=1-2.

Thus, the inventors note that a highly favourable amino acid variationwhich would be selected preferentially is not observed. As discussedabove, two variations which were prominently represented in Table 1 ofscFv variants were analysed closer. Any scFv variant in Table 1 witheither of these mutations along with one or more further mutationsdisplayed a potency which was at least as improved as a clone containingany one of these two single amino acid variations in the parentBAK278D6. There is therefore no evidence that a highly favourable aminoacid variation, that would allow scenario A4, is present in the panel.

This observation led the inventors to conclude that there were nonon-favourable mutations present in this set of scFv variants. Thismeans scenarios A4 and B1 to B3 are not relevant and the hypothesis isvalid.

Accordingly, as noted already, the present invention provides specificbinding members comprising the defined sets of CDR's, in particular theset of CDR's of BAK278D6, and sets of CDR's of the BAK278D6 lineage,with one or two substitutions within the set of CDR's, e.g. the BAK502G9set of CDR's.

The relevant set of CDR's is provided within antibody framework regionsor other protein scaffold, e.g. fibronectin or cytochrome B [115, 116].Preferably antibody framework regions are employed, and where they areemployed they are preferably germline, more preferably the antibodyframework region for the heavy chain may be DP14 from the VH1 family.The preferred framework region for the light chain may beλ3-3H. For theBAK502G9 set of CDR's it is preferred that the antibody frameworkregions are for VH FR1, SEQ ID NO: 27, for

VH FR2, SEQ ID NO: 28, for VH FR3, SEQ ID NO 29, for light chain FR1,SEQ ID NO: 30, for light chain FR2, SEQ ID NO: 31, for light chain FR3,SEQ ID NO: 32. In a highly preferred embodiment, a VH domain is providedwith the amino acid sequence of SEQ ID NO: 15, this being termed“BAK502G9 VH domain”. In a further highly preferred embodiment, a VLdomain is provided with the amino acid sequence of SEQ ID NO: 16, thisbeing termed “BAK502G9 VL domain”. A highly preferred antibodyantigen-binding site provided in accordance with the present inventionis composed of the BAK502G9 VH domain, SEQ ID NO: 15, and the BAK502G9VL domain, SEQ ID NO: 16. This antibody antigen-binding site may beprovided within any desired antibody molecule format, e.g. scFv, Fab,IgG, IgG4, dAb etc., as is discussed further elsewhere herein.

In a further highly preferred embodiment, the present invention providesan IgG4 antibody molecule comprising the BAK502G9 VH domain, SEQ ID NO:15, and the BAK502G9 VL domain, SEQ ID NO: 16. This is termed herein“BAK502G9 IgG4”.

Other IgG4 or other antibody molecules comprising the BAK502G9 VHdomain, SEQ ID NO: 15, and/or the BAK502G9 VL domain, SEQ ID NO: 16, areprovided by the present invention, as are other antibody moleculescomprising the BAK502G9 set of HCDR's (SEQ ID NO: 7, 8 and 9) within anantibody VH domain, and/or the BAK502G9 set of LCDR's (SEQ ID NO: 10, 11and 12) within an antibody VL domain.

It is convenient to point out here that “and/or” where used herein is tobe taken as specific disclosure of each of the two specified features orcomponents with or without the other. For example “A and/or B” is to betaken as specific disclosure of each of (i) A, (ii) B and (iii) A and B,just as if each is set out individually herein.

As noted, the present invention provides a specific binding member whichbinds human IL-13 and which comprises the BAK502G9 VH domain (SEQ ID NO:15) and/or the BAK502G9 VL domain (SEQ ID NO: 16).

Generally, a VH domain is paired with a VL domain to provide an antibodyantigen binding site, although as discussed further below a VH domainalone may be used to bind antigen. In one preferred embodiment, theBAK502G9 VH domain (SEQ ID NO: 15) is paired with the BAK502G9 VL domain(SEQ ID NO: 16), so that an antibody antigen binding site is formedcomprising both the BAK502G9 VH and VL domains. In other embodiments,the BAK502G9 VH is paired with a VL domain other than the BAK502G9 VL.Light-chain promiscuity is well established in the art.

Similarly, any set of HCDR's of the BAK278D6 lineage can be provided ina VH domain that is used as a specific binding member alone or incombination with a VL domain. A VH domain may be provided with a set ofHCDR's of a BAK278D6 lineage antibody, e.g. as shown in Table 1, and ifsuch a VH domain is paired with a VL domain, then the VL domain may beprovided with a set of LCDR's of a BAK278D6 lineage antibody, e.g. asshown in Table 1. A pairing of a set of HCDR's and a set of LCDR's maybe as shown in Table 1, providing an antibody antigen-binding sitecomprising a set of CDR's as shown in Table 1. The framework regions ofthe VH and/or VL domains may be germline frameworks. Frameworks regionsof the heavy chain domain may be selected from the VH-1 family, and apreferred VH-1 framework is DP-14 framework. Framework regions of thelight chain may be selected from the λ3 family, and a preferred suchframework is λ3 3H.

One or more CDRs may be taken from the BAK502G9 VH or VL domain andincorporated into a suitable framework. This is discussed furtherherein. BAK502G9 HCDR's 1, 2 and 3 are shown in SEQ ID NO: 7, 8 and 9,respectively. BAK502G9 LCDR's 1, 2 and 3 are shown in SEQ ID NO: 10, 11and 12, respectively.

The same applies for other BAK278D6 lineage CDR's and sets of CDR's asshown in Table 1.

Further embodiments of the invention relate to a specific binding membercomprising the VH and/or VL domain, or an antigen binding sitecomprising CDRs of the VH and/or VL domain of the antibody moleculedisclosed herein as 167A11 (VH: SEQ ID NO: 23 and VL: SEQ ID NO: 24) andits derivatives 615E3 (VH:SEQ ID NO: 33 and VL: SEQ ID NO: 34) BAK582F7(VH CDR's SEQ ID's 141-143) and BAK612B5 (VH CDR's SEQ ID's 147-149).These recognise human IL-13. The derivatives of 167A11 from VH CDR3randomisation are potent scFv molecules (5-6 nM). The 167A11 lineage maybe employed in any aspect and embodiment of the present invention asdisclosed herein for other molecules, for instance methods of mutationand selection of antigen binding sites with improved potency.

Variants of the VH and VL domains and CDRs of the present invention,including those for which amino acid sequences are set out herein, andwhich can be employed in specific binding members for IL-13 can beobtained by means of methods of sequence alteration or mutation andscreening. Such methods are also provided by the present invention.

Variable domain amino acid sequence variants of any of the VH and VLdomains whose sequences are specifically disclosed herein may beemployed in accordance with the present invention, as discussed.Particular variants may include one or more amino acid sequencealterations (addition, deletion, substitution and/or insertion of anamino acid residue), may be less than about 20 alterations, less thanabout 15 alterations, less than about 10 alterations or less than about5 alterations, 4, 3, 2 or 1. Alterations may be made in one or moreframework regions and/or one or more CDR's.

In accordance with further aspects of the present invention there isprovided a specific binding member which competes for binding to antigenwith any specific binding member which both binds the antigen andcomprises a specific binding member, VH and/or VL domain disclosedherein, or HCDR3 disclosed herein, or variant of any of these.Competition between binding members may be assayed easily in vitro, forexample using ELISA and/or by tagging a specific reporter molecule toone binding member which can be detected in the presence of otheruntagged binding member(s), to enable identification of specific bindingmembers which bind the same epitope or an overlapping epitope.

Thus, a further aspect of the present invention provides a specificbinding member comprising a human antibody antigen-binding site whichcompetes with a BAK502G9 antibody molecule, in particular BAK502G9 scFvand/or IgG4, for binding to IL-13. In further aspects the presentinvention provides a specific binding member comprising a human antibodyantigen-binding site which competes with an antibody antigen-bindingsite for binding to IL-13, wherein the antibody antigen-binding site iscomposed of a VH domain and a VL domain, and wherein the VH and VLdomains comprise a set of CDR's of the BAK278D6 lineage.

Various methods are available in the art for obtaining antibodiesagainst IL-13 and which may compete with a BAK502G9 antibody molecule,an antibody molecule with a BAK502G9 set of

CDR's, or an antibody molecule with a set of CDR's of BAK278D6 lineage,for binding to IL-13.

In a further aspect, the present invention provides a method ofobtaining one or more specific binding members able to bind the antigen,the method including bringing into contact a library of specific bindingmembers according to the invention and said antigen, and selecting oneor more specific binding members of the library able to bind saidantigen.

The library may be displayed on the surface of bacteriophage particles,each particle containing nucleic acid encoding the antibody VH variabledomain displayed on its surface, and optionally also a displayed VLdomain if present.

Following selection of specific binding members able to bind the antigenand displayed on bacteriophage particles, nucleic acid may be taken froma bacteriophage particle displaying a said selected specific bindingmember. Such nucleic acid may be used in subsequent production of aspecific binding member or an antibody VH variable domain (optionally anantibody VL variable domain) by expression from nucleic acid with thesequence of nucleic acid taken from a bacteriophage particle displayinga said selected specific binding member.

An antibody VH variable domain with the amino acid sequence of anantibody VH variable domain of a said selected specific binding membermay be provided in isolated form, as may a specific binding membercomprising such a VH domain. Ability to bind IL-13 may be furthertested, also ability to compete with BAK502G9 (e.g. in scFv formatand/or IgG format, e.g. IgG4) for binding to IL-13. Ability toneutralise IL-13 may be tested, as discussed further below.

A specific binding member according to the present invention may bindIL-13 with the affinity of a BAK502G9 antibody molecule, e.g. scFv, orpreferably BAK502G9 IgG4, or with an affinity that is better.

A specific binding member according to the present invention mayneutralise IL-13 with the potency of a BAK502G9 antibody molecule, e.g.scFv, or preferably BAK502G9 IgG4, or with a potency that is better.

A specific binding member according to the present invention mayneutralise naturally occurring IL-13 with the potency of a BAK502G9antibody molecule, e.g. scFv, or preferably BAK502G9 IgG4, or with apotency that is better.

Binding affinity and neutralisation potency of different specificbinding members can be compared under appropriate conditions.

The antibodies of the present invention have a number of advantages overexisting commercial anti-IL-13 antibodies, in particular threecommercial rodent anti-human IL-13 antibodies namely, JES10-5A2(BioSource), B-B13 (Euroclone) and clone 321166 (R&D Systems). Thepotency of the antibodies of the present invention was compared withcommercial antibodies JES10-A2 and B-B13. Clone 321166 was not evaluatedas previous experiments revealed that this clone was considerably lesspotent than other known commercial antibodies.

The efficacy and use of the rodent commercial IL-13 antibodies in man islikely to be limited, because of their increased potential to induceimmunogenic responses and therefore more rapid clearance from the body.Kinetic analysis of the antibodies of the present invention in non-humanprimates suggests that these antibodies have a clearance rate which issimilar to that of other known human or humanised antibodies.

Antibodies provided by various embodiments of the present inventionrecognize non-human primate IL-13, including rhesus and cynomolgusIL-13. Determining efficacy and safety profiles of an antibody innon-human primates is extremely valuable as it provides a means forpredicting the antibody's safety, pharmacokinetic and pharmacodynamicprofile in humans.

Moreover, antibodies of various embodiments of the present inventionfurther recognize the human IL-13 variant, Q130R, which is associatedwith asthma. Cross reactivity with variant IL-13 allows antibodies ofthe present invention and compositions comprising antibodies of thepresent invention to be used for the treatment of patients withwild-type and variant IL-13.

A preferred embodiment of the present invention comprises antibodiesthat neutralise naturally occurring IL-13 with a potency that is equalto or better than the potency of a IL-13 antigen binding site formed byBAK502G9 VH domain (SEQ ID NO:15) and the BAK502G9 VL domain (SEQ ID NO:16). For example, the inventors have demonstrated that representativeclones such as BAK502G9, 1167F2 and 1183H4 are significantly more potentagainst naturally occurring IL-13 than known commercial antibodies (FIG.7).

In addition to antibody sequences, a specific binding member accordingto the present invention may comprise other amino acids, e.g. forming apeptide or polypeptide, such as a folded domain, or to impart to themolecule another functional characteristic in addition to ability tobind antigen. Specific binding members of the invention may carry adetectable label, or may be conjugated to a toxin or a targeting moietyor enzyme (e.g. via a peptidyl bond or linker).

In further aspects, the invention provides an isolated nucleic acidwhich comprises a sequence encoding a specific binding member, VH domainand/or VL domains according to the present invention, and methods ofpreparing a specific binding member, a VH domain and/or a VL domain ofthe invention, which comprise expressing said nucleic acid underconditions to bring about production of said specific binding member, VHdomain and/or VL domain, and recovering it.

Specific binding members according to the invention may be used in amethod of treatment or diagnosis of the human or animal body, such as amethod of treatment (which may include prophylactic treatment) of adisease or disorder in a human patient which comprises administering tosaid patient an effective amount of a specific binding member of theinvention. Conditions treatable in accordance with the present inventioninclude any in which IL-13 plays a role, especially asthma, atopicdermatitis, allergic rhinitis, fibrosis, chronic obstructive pulmonarydisease, scleroderma, inflammatory bowel disease and Hodgkin's lymphoma.Further, the antibodies of the present invention may also be used intreating tumours and viral infections as these antibodies will inhibitIL-13 mediated immunosupression [64, 65].

A further aspect of the present invention provides nucleic acid,generally isolated, encoding an antibody VH variable domain and/or VLvariable domain disclosed herein.

Another aspect of the present invention provides nucleic acid, generallyisolated, encoding a VH CDR or VL CDR sequence disclosed herein,especially a VH CDR selected from SEQ ID NO's: 7, 8 and 9 or a VL CDRselected from SEQ ID NO's: 10, 11 and 12, most preferably BAK502G9 VHCDR3 (SEQ ID NO: 9). Nucleic acid encoding the BAK502G9 set of CDR's,nucleic acid encoding the BAK502G9 set of HCDR's and nucleic acidencoding the BAK502G9 set of LCDR's are also provided by the presentinvention, as are nucleic acids encoding individual CDR's, HCDR's,LCDR's and sets of CDR's, HCDR's, LCDR's of the BAK278D6 lineage.

A further aspect provides a host cell transformed with nucleic acid ofthe invention.

A yet further aspect provides a method of production of an antibody VHvariable domain, the method including causing expression from encodingnucleic acid. Such a method may comprise culturing host cells underconditions for production of said antibody VH variable domain.

Analogous methods for production of VL variable domains and specificbinding members comprising a VH and/or VL domain are provided as furtheraspects of the present invention.

A method of production may comprise a step of isolation and/orpurification of the product.

A method of production may comprise formulating the product into acomposition including at least one additional component, such as apharmaceutically acceptable excipient.

These and other aspects of the invention are described in further detailbelow.

Terminology

Specific Binding Member

This describes a member of a pair of molecules which have bindingspecificity for one another. The members of a specific binding pair maybe naturally derived or wholly or partially synthetically produced. Onemember of the pair of molecules has an area on its surface, or a cavity,which specifically binds to and is therefore complementary to aparticular spatial and polar organisation of the other member of thepair of molecules. Thus the members of the pair have the property ofbinding specifically to each other. Examples of types of specificbinding pairs are antigen-antibody, biotin-avidin, hormone-hormonereceptor, receptor-ligand, enzyme-substrate. The present invention isconcerned with antigen-antibody type reactions.

Antibody Molecule

This describes an immunoglobulin whether natural or partly or whollysynthetically produced. The term also covers any polypeptide or proteincomprising an antibody binding domain. Antibody fragments which comprisean antigen binding domain are molecules such as Fab, scFv, Fv, dAb, Fd;and diabodies.

It is possible to take monoclonal and other antibodies and usetechniques of recombinant DNA technology to produce other antibodies orchimeric molecules which retain the specificity of the originalantibody. Such techniques may involve introducing DNA encoding theimmunoglobulin variable region, or the complementarity determiningregions (CDRs), of an antibody to the constant regions, or constantregions plus framework regions, of a different immunoglobulin. See, forinstance, EP-A-184187, GB 2188638A or EP-A-239400, and a large body ofsubsequent literature. A hybridoma or other cell producing an antibodymay be subject to genetic mutation or other changes, which may or maynot alter the binding specificity of antibodies produced.

As antibodies can be modified in a number of ways, the term “antibodymolecule” should be construed as covering any specific binding member orsubstance having an antibody antigen-binding domain with the requiredspecificity. Thus, this term covers antibody fragments and derivatives,including any polypeptide comprising an immunoglobulin binding domain,whether natural or wholly or partially synthetic. Chimeric moleculescomprising an immunoglobulin binding domain, or equivalent, fused toanother polypeptide are therefore included. Cloning and expression ofchimeric antibodies are described in EP-A-0120694 and EP-A-0125023, anda large body of subsequent literature.

Further techniques available in the art of antibody engineering havemade it possible to isolate human and humanised antibodies. For example,human hybridomas can be made as described by Kontermann et al [107].Phage display, another established technique for generating specificbinding members has been described in detail in many publications suchas Kontermann et al [107] and WO92/01047 (discussed further below).Transgenic mice in which the mouse antibody genes are inactivated andfunctionally replaced with human antibody genes while leaving intactother components of the mouse immune system, can be used for isolatinghuman antibodies to human antigens [108].

Synthetic antibody molecules may be created by expression from genesgenerated by means of oligonucleotides synthesized and assembled withinsuitable expression vectors, for example as described by Knappik et al.J. Mol. Biol. (2000) 296, 57-86 or Krebs et al. Journal of ImmunologicalMethods 254 2001 67-84.

It has been shown that fragments of a whole antibody can perform thefunction of binding antigens. Examples of binding fragments are (i) theFab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fdfragment consisting of the VH and CH1 domains; (iii) the Fv fragmentconsisting of the VL and VH domains of a single antibody; (iv) the dAbfragment (Ward, E.S. et al., Nature 341, 544-546 (1989), McCafferty etal (1990) Nature, 348, 552-554) which consists of a VH domain; (v)isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragmentcomprising two linked Fab fragments (vii) single chain Fv molecules(scFv), wherein a VH domain and a VL domain are linked by a peptidelinker which allows the two domains to associate to form an antigenbinding site (Bird et al, Science, 242, 423-426, 1988; Huston et al,PNAS USA, 85, 5879-5883, 1988); (viii) bispecific single chain Fv dimers(PCT/US92/09965) and (ix) “diabodies”, multivalent or multispecificfragments constructed by gene fusion (WO94/13804; P. Holliger et al,Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993). Fv, scFv or diabodymolecules may be stabilised by the incorporation of disulphide bridgeslinking the VH and VL domains (Y. Reiter et al, Nature Biotech, 14,1239-1245, 1996). Minibodies comprising a scFv joined to a CH3 domainmay also be made (S. Hu et al, Cancer Res., 56, 3055-3061, 1996).

Where bispecific antibodies are to be used, these may be conventionalbispecific antibodies, which can be manufactured in a variety of ways(Holliger, P. and Winter G. Current Opinion Biotechnol. 4, 446-449(1993)), e.g. prepared chemically or from hybrid hybridomas, or may beany of the bispecific antibody fragments mentioned above. Examples ofbispecific antibodies include those of the BiTE™ technology in which thebinding domains of two antibodies with different specificity can be usedand directly linked via short flexible peptides. This combines twoantibodies on a short single polypeptide chain. Diabodies and scFv canbe constructed without an Fc region, using only variable domains,potentially reducing the effects of anti-idiotypic reaction.

Bispecific diabodies, as opposed to bispecific whole antibodies, mayalso be particularly useful because they can be readily constructed andexpressed in E.coli. Diabodies (and many other polypeptides such asantibody fragments) of appropriate binding specificities can be readilyselected using phage display (WO94/13804) from libraries. If one arm ofthe diabody is to be kept constant, for instance, with a specificitydirected against IL-13, then a library can be made where the other armis varied and an antibody of appropriate specificity selected.Bispecific whole antibodies may be made by knobs-into-holes engineering(J. B. B. Ridgeway et al, Protein Eng., 9, 616-621, 1996).

Antigen-Binding Domain

This describes the part of an antibody molecule which comprises the areawhich specifically binds to and is complementary to part or all of anantigen. Where an antigen is large, an antibody may only bind to aparticular part of the antigen, which part is termed an epitope. Anantigen binding domain may be provided by one or more antibody variabledomains (e.g. a so-called Fd antibody fragment consisting of a VHdomain). Preferably, an antigen binding domain comprises an antibodylight chain variable region (VL) and an antibody heavy chain variableregion (VH).

Specific

This may be used to refer to the situation in which one member of aspecific binding pair will not show any significant binding to moleculesother than its specific binding partner(s). The term is also applicablewhere e.g. an antigen binding domain is specific for a particularepitope which is carried by a number of antigens, in which case thespecific binding member carrying the antigen binding domain will be ableto bind to the various antigens carrying the epitope.

Comprise

This is generally used in the sense of include, that is to saypermitting the presence of one or more features or components.

Isolated

This refers to the state in which specific binding members of theinvention, or nucleic acid encoding such binding members, will generallybe in accordance with the present invention. Isolated members andisolated nucleic acid will be free or substantially free of materialwith which they are naturally associated such as other polypeptides ornucleic acids with which they are found in their natural environment, orthe environment in which they are prepared (e.g. cell culture) when suchpreparation is by recombinant DNA technology practised in vitro or invivo. Members and nucleic acid may be formulated with diluents oradjuvants and still for practical purposes be isolated—for example themembers will normally be mixed with gelatin or other carriers if used tocoat microtitre plates for use in immunoassays, or will be mixed withpharmaceutically acceptable carriers or diluents when used in diagnosisor therapy. Specific binding members may be glycosylated, eithernaturally or by systems of heterologous eukaryotic cells (e.g. CHO orNS0 (ECACC 85110503) cells, or they may be (for example if produced byexpression in a prokaryotic cell) unglycosylated.

Naturally Occurring IL-13

This generally refers to a state in which the IL-13 protein or fragmentsthereof may occur. Naturally occurring IL-13 means IL-13 protein whichis naturally produced by a cell, without prior introduction of encodingnucleic acid using recombinant technology. Thus, naturally occurringIL-13 may be as produced naturally by for example CD4+ T cells and/or asisolated from a mammal, e.g. human, non-human primate, rodent such asrat or mouse.

Recombinant IL-13

This refers to a state in which the IL-13 protein or fragments thereofmay occur. Recombinant IL-13 means IL-13 protein or fragments thereofproduced by recombinant DNA in a heterologous host. Recombinant IL-13may differ from naturally occurring IL-13 by glycosylation.

Recombinant proteins expressed in prokaryotic bacterial expressionsystems are not glycosylated while those expressed in eukaryotic systemssuch as mammalian or insect cells are glycosylated. Proteins expressedin insect cells however differ in glycosylation from proteins expressedin mammalian cells.

By “substantially as set out” it is meant that the relevant CDR or VH orVL domain of the invention will be either identical or highly similar tothe specified regions of which the sequence is set out herein. By“highly similar” it is contemplated that from 1 to 5, preferably from 1to 4 such as 1 to 3 or 1 or 2, or 3 or 4, amino acid substitutions maybe made in the CDR and/or VH or VL domain.

The structure for carrying a CDR or a set of CDR's of the invention willgenerally be of an antibody heavy or light chain sequence or substantialportion thereof in which the CDR or set of CDR's is located at alocation corresponding to the CDR or set of CDR's of naturally occurringVH and VL antibody variable domains encoded by rearranged immunoglobulingenes. The structures and locations of immunoglobulin variable domainsmay be determined by reference to (Kabat, E. A. et al, Sequences ofProteins of Immunological Interest. 4th Edition. US Department of Healthand Human Services. 1987, and updates thereof, now available on theInternet http://immuno.bme.nwu.edu or find “Kabat” using any searchengine).

CDR's can also be carried by other scaffolds such as fibronectin orcytochrome B [115, 116].

Preferably, a CDR amino acid sequence substantially as set out herein iscarried as a CDR in a human variable domain or a substantial portionthereof. The HCDR3 sequences substantially as set out herein representpreferred embodiments of the present invention and it is preferred thateach of these is carried as a HCDR3 in a human heavy chain variabledomain or a substantial portion thereof.

Variable domains employed in the invention may be obtained from anygerm-line or rearranged human variable domain, or may be a syntheticvariable domain based on consensus sequences of known human variabledomains. A CDR sequence of the invention (e.g. CDR3) may be introducedinto a repertoire of variable domains lacking a CDR (e.g. CDR3), usingrecombinant DNA technology.

For example, Marks et al (Bio/Technology, 1992, 10:779-783) describemethods of producing repertoires of antibody variable domains in whichconsensus primers directed at or adjacent to the 5′ end of the variabledomain area are used in conjunction with consensus primers to the thirdframework region of human VH genes to provide a repertoire of VHvariable domains lacking a CDR3. Marks et al further describe how thisrepertoire may be combined with a CDR3 of a particular antibody. Usinganalogous techniques, the CDR3-derived sequences of the presentinvention may be shuffled with repertoires of VH or VL domains lacking aCDR3, and the shuffled complete VH or VL domains combined with a cognateVL or VH domain to provide specific binding members of the invention.The repertoire may then be displayed in a suitable host system such asthe phage display system of WO92/01047 or any of a subsequent large bodyof literature, including Kay, B. K., Winter, J., and McCafferty, J.(1996) Phage Display of Peptides and Proteins: A Laboratory Manual, SanDiego: Academic Press, so that suitable specific binding members may beselected. A repertoire may consist of from anything from 10⁴ individualmembers upwards, for example from 10⁶ to 10⁸ or 10¹° members. Othersuitable host systems include yeast display, bacterial display, T7display, ribosome display and so on. For a review of ribosome displayfor see Lowe D and Jermutus L, 2004, Curr. Pharm, Biotech, 517-27, alsoWO92/01047.

Analogous shuffling or combinatorial techniques are also disclosed byStemmer (Nature, 1994, 370:389-391), who describes the technique inrelation to a β-lactamase gene but observes that the approach may beused for the generation of antibodies.

A further alternative is to generate novel VH or VL regions carryingCDR-derived sequences of the invention using random mutagenesis of oneor more selected VH and/or VL genes to generate mutations within theentire variable domain. Such a technique is described by Gram et al(1992, Proc. Natl. Acad. Sci., USA, 89:3576-3580), who used error-pronePCR. In preferred embodiments one or two amino acid substitutions aremade within a set of HCDR's and/or LCDR's.

Another method which may be used is to direct mutagenesis to CDR regionsof VH or VL genes. Such techniques are disclosed by Barbas et al, (1994,Proc. Natl. Acad. Sci., USA, 91:3809-3813) and Schier et al (1996, J.Mol. Biol. 263:551-567).

All the above described techniques are known as such in the art and inthemselves do not form part of the present invention. The skilled personwill be able to use such techniques to provide specific binding membersof the invention using routine methodology in the art.

A further aspect of the invention provides a method for obtaining anantibody antigen binding domain specific for IL-13 antigen, the methodcomprising providing by way of addition, deletion, substitution orinsertion of one or more amino acids in the amino acid sequence of a VHdomain set out herein a VH domain which is an amino acid sequencevariant of the VH domain, optionally combining the VH domain thusprovided with one or more VL domains, and testing the VH domain or VH/VLcombination or combinations to identify a specific binding member or anantibody antigen binding domain specific for IL-13 antigen andoptionally with one or more preferred properties, preferably ability toneutralise IL-13 activity. Said VL domain may have an amino acidsequence which is substantially as set out herein.

An analogous method may be employed in which one or more sequencevariants of a VL domain disclosed herein are combined with one or moreVH domains.

In a preferred embodiment, BAK502G9 VH domain (SEQ ID NO: 15) may besubject to mutation to provide one or more VH domain amino acid sequencevariants, and/or BAK502G9 VL (SEQ ID NO: 16).

A further aspect of the invention provides a method of preparing aspecific binding member specific for IL-13 antigen, which methodcomprises:

-   -   (a) providing a starting repertoire of nucleic acids encoding a        VH domain which either include a CDR3 to be replaced or lack a        CDR3 encoding region;    -   (b) combining said repertoire with a donor nucleic acid encoding        an amino acid sequence substantially as set out herein for a VH        CDR3 such that said donor nucleic acid is inserted into the CDR3        region in the repertoire, so as to provide a product repertoire        of nucleic acids encoding a VH domain;    -   (c) expressing the nucleic acids of said product repertoire;    -   (d) selecting a specific binding member specific for a IL-13;        and    -   (e) recovering said specific binding member or nucleic acid        encoding it.

Again, an analogous method may be employed in which a VL CDR3 of theinvention is combined with a repertoire of nucleic acids encoding a VLdomain which either include a CDR3 to be replaced or lack a CDR3encoding region.

Similarly, one or more, or all three CDRs may be grafted into arepertoire of VH or VL domains which are then screened for a specificbinding member or specific binding members specific for IL-13.

In a preferred embodiment, one or more of BAK502G9 HCDR1 (SEQ ID NO: 7),HCDR2 (SEQ ID NO: 8) and HCDR3 (SEQ ID NO: 9), or the BAK502G9 set ofHCDR's, may be employed, and/or one or more of BAK502G9 LCDR1 (SEQ IDNO: 10), LCDR2 (SEQ ID NO: 11), or the BAK502G9 set of LCDR's.

A substantial portion of an immunoglobulin variable domain will compriseat least the three CDR regions, together with their interveningframework regions. Preferably, the portion will also include at leastabout 50% of either or both of the first and fourth framework regions,the 50% being the C-terminal 50% of the first framework region and theN-terminal 50% of the fourth framework region. Additional residues atthe N-terminal or C-terminal end of the substantial part of the variabledomain may be those not normally associated with naturally occurringvariable domain regions. For example, construction of specific bindingmembers of the present invention made by recombinant DNA techniques mayresult in the introduction of N- or C-terminal residues encoded bylinkers introduced to facilitate cloning or other manipulation steps.Other manipulation steps include the introduction of linkers to joinvariable domains of the invention to further protein sequences includingimmunoglobulin heavy chains, other variable domains (for example in theproduction of diabodies) or protein labels as discussed in more detailelsewhere herein.

Although in a preferred aspect of the invention specific binding memberscomprising a pair of VH and VL domains are preferred, single bindingdomains based on either VH or VL domain sequences form further aspectsof the invention. It is known that single immunoglobulin domains,especially VH domains, are capable of binding target antigens in aspecific manner.

In the case of either of the single specific binding domains, thesedomains may be used to screen for complementary domains capable offorming a two-domain specific binding member able to bind IL-13.

This may be achieved by phage display screening methods using theso-called hierarchical dual combinatorial approach as disclosed inWO92/01047, in which an individual colony containing either an H or Lchain clone is used to infect a complete library of clones encoding theother chain (L or H) and the resulting two-chain specific binding memberis selected in accordance with phage display techniques such as thosedescribed in that reference. This technique is also disclosed in Markset al, ibid.

Specific binding members of the present invention may further compriseantibody constant regions or parts thereof. For example, a VL domain maybe attached at its C-terminal end to antibody light chain constantdomains including human Cκ or Cλ chains, preferably Cλ chains.Similarly, a specific binding member based on a VH domain may beattached at its C-terminal end to all or part (e.g. a CH1 domain) of animmunoglobulin heavy chain derived from any antibody isotype, e.g. IgG,IgA, IgE and IgM and any of the isotype sub-classes, particularly IgG1and IgG4. IgG4 is preferred. IgG4 is preferred because it does not bindcomplement and does not create effector functions. Any synthetic orother constant region variant that has these properties and stabilizesvariable regions is also preferred for use in embodiments of the presentinvention.

Specific binding members of the invention may be labelled with adetectable or functional label. Detectable labels include radiolabelssuch as ¹³¹I or ⁹⁹Tc, which may be attached to antibodies of theinvention using conventional chemistry known in the art of antibodyimaging. Labels also include enzyme labels such as horseradishperoxidase. Labels further include chemical moieties such as biotinwhich may be detected via binding to a specific cognate detectablemoiety, e.g. labelled avidin.

Specific binding members of the present invention are designed to beused in methods of diagnosis or treatment in human or animal subjects,preferably human.

Accordingly, further aspects of the invention provide methods oftreatment comprising administration of a specific binding member asprovided, pharmaceutical compositions comprising such a specific bindingmember, and use of such a specific binding member in the manufacture ofa medicament for administration, for example in a method of making amedicament or pharmaceutical composition comprising formulating thespecific binding member with a pharmaceutically acceptable excipient.

Clinical indications in which an anti-IL-13 antibody may be used toprovide therapeutic benefit include asthma, atopic dermatitis, allergicrhinitis, fibrosis, chronic obstructive pulmonary disease, inflammatorybowel disease, scleroderma and Hodgkin's lymphoma . As alreadyexplained, anti-IL-13 treatment is effective for all these diseases.

Anti-IL-13 treatment may be given orally, by injection (for example,subcutaneously, intravenously, intraperitoneal or intramuscularly), byinhalation, or topically (for example intraocular, intranasal, rectal,into wounds, on skin). The route of administration can be determined bythe physicochemical characteristics of the treatment, by specialconsiderations for the disease or by the requirement to optimiseefficacy or to minimise side-effects.

It is envisaged that anti-IL-13 treatment will not be restricted to usein the clinic. Therefore, subcutaneous injection using a needle freedevice is also preferred.

Combination treatments may be used to provide significant synergisticeffects, particularly the combination of an anti-IL-13 specific bindingmember with one or more other drugs. A specific binding member accordingto the present invention may be provided in combination or addition toshort or long acting beta agonists, corticosteroids, cromoglycate,leukotriene (receptor) antagonists, methyl xanthines and theirderivatives, IL-4 inhibitors, muscarinic receptor antagonists, IgEinhibitors, histaminic inhibitors, IL-5 inhibitors, eotaxin/CCR3inhibitors, PDE4 inhibitors, TGF-beta antagonists, interferon-gamma,perfenidone, chemotherapeutic agents and immunotherapeutic agents.

Combination treatment with one or more short or long acting betaagonists, corticosteroids, cromoglycate, leukotriene (receptor)antagonists, xanthines, IgE inhibitors, IL-4 inhibitors, IL-5inhibitors, eotaxin/CCR3 inhibitors, PDE4 inhibitors may be employed fortreatment of asthma. Antibodies of the present invention can also beused in combination with corticosteroids, anti-metabolites, antagonistsof TGF-beta and its downstream signalling pathway, for treatment offibrosis. Combination therapy of these antibodies with PDE4 inhibitors,xanthines and their derivatives, muscarinic receptor antagonists, shortand long beta antagonists can be useful for treating chronic obstructivepulmonary disease. Similar consideration of combinations apply to theuse of anti-IL-13 treatment for atopic dermatitis, allergic rhinitis,chronic obstructive pulmonary disease, inflammatory bowel disease,scleroderma and Hodgkin's lymphoma.

In accordance with the present invention, compositions provided may beadministered to individuals. Administration is preferably in a“therapeutically effective amount”, this being sufficient to showbenefit to a patient. Such benefit may be at least amelioration of atleast one symptom. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofwhat is being treated. Prescription of treatment, e.g. decisions ondosage etc, is within the responsibility of general practitioners andother medical doctors. Appropriate doses of antibody are well known inthe art; see Ledermann J.A. et al. (1991) Int. J. Cancer 47: 659-664;Bagshawe K. D. et al. (1991) Antibody, Immunoconjugates andRadiopharmaceuticals 4: 915-922.

The precise dose will depend upon a number of factors, including whetherthe antibody is for diagnosis or for treatment, the size and location ofthe area to be treated, the precise nature of the antibody (e.g. wholeantibody, fragment or diabody), and the nature of any detectable labelor other molecule attached to the antibody. A typical antibody dose willbe in the range 100 μg to 1 gm for systemic applications, and 1 μg to 1mg for topical applications. Typically, the antibody will be a wholeantibody, preferably the IgG4 isotype. This is a dose for a singletreatment of an adult patient, which may be proportionally adjusted forchildren and infants, and also adjusted for other antibody formats inproportion to molecular weight. Treatments may be repeated at daily,twice-weekly, weekly or monthly intervals, at the discretion of thephysician. In preferred embodiments of the present invention, treatmentis periodic, and the period between administrations is about two weeksor more, preferably about three weeks or more, more preferably aboutfour weeks or more, or about once a month.

Specific binding members of the present invention will usually beadministered in the form of a pharmaceutical composition, which maycomprise at least one component in addition to the specific bindingmember.

Thus pharmaceutical compositions according to the present invention, andfor use in accordance with the present invention, may comprise, inaddition to active ingredient, a pharmaceutically acceptable excipient,carrier, buffer, stabiliser or other materials well known to thoseskilled in the art. Such materials should be non-toxic and should notinterfere with the efficacy of the active ingredient. The precise natureof the carrier or other material will depend on the route ofadministration, which may be oral, or by injection, e.g. intravenous.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may comprise a solid carriersuch as gelatin or an adjuvant. Liquid pharmaceutical compositionsgenerally comprise a liquid carrier such as water, petroleum, animal orvegetable oils, mineral oil or synthetic oil. Physiological salinesolution, dextrose or other saccharide solution or glycols such asethylene glycol, propylene glycol or polyethylene glycol may beincluded.

For intravenous injection, or injection at the site of affliction, theactive ingredient will be in the form of a parenterally acceptableaqueous solution which is pyrogen-free and has suitable pH, isotonicityand stability. Those of relevant skill in the art are well able toprepare suitable solutions using, for example, isotonic vehicles such asSodium Chloride Injection, Ringer's Injection, Lactated Ringer'sInjection. Preservatives, stabilisers, buffers, antioxidants and/orother additives may be included, as required.

A composition may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated.

Specific binding members of the present invention may be formulated inliquid or solid forms depending on the physicochemical properties of themolecule and the route of delivery. Formulations may include excipients,or combinations of excipients, for example: sugars, amino acids andsurfactants. Liquid formulations may include a wide range of antibodyconcentrations and pH. Solid formulations may be produced bylyophilisation, spray drying, or drying by supercritical fluidtechnology, for example. Formulations of anti-IL-13 will depend upon theintended route of delivery: for example, formulations for pulmonarydelivery may consist of particles with physical properties that ensurepenetration into the deep lung upon inhalation; topical formulations mayinclude viscosity modifying agents, which prolong the time that the drugis resident at the site of action.

The present invention provides a method comprising causing or allowingbinding of a specific binding member as provided herein to IL-13. Asnoted, such binding may take place in vivo, e.g. followingadministration of a specific binding member, or nucleic acid encoding aspecific binding member, or it may take place in vitro, for example inELISA, Western blotting, immunocytochemistry, immuno-precipitation,affinity chromatography, or cell based assays such as a TF-1 assay.

The amount of binding of specific binding member to IL-13 may bedetermined. Quantitation may be related to the amount of the antigen ina test sample, which may be of diagnostic interest.

A kit comprising a specific binding member or antibody moleculeaccording to any aspect or embodiment of the present invention is alsoprovided as an aspect of the present invention. In a kit of theinvention, the specific binding member or antibody molecule may belabelled to allow its reactivity in a sample to be determined, e.g. asdescribed further below. Components of a kit are generally sterile andin sealed vials or other containers. Kits may be employed in diagnosticanalysis or other methods for which antibody molecules are useful. A kitmay contain instructions for use of the components in a method, e.g. amethod in accordance with the present invention. Ancillary materials toassist in or to enable performing such a method may be included within akit of the invention.

The reactivities of antibodies in a sample may be determined by anyappropriate means. Radioimmunoassay (RIA) is one possibility.Radioactive labelled antigen is mixed with unlabelled antigen (the testsample) and allowed to bind to the antibody. Bound antigen is physicallyseparated from unbound antigen and the amount of radioactive antigenbound to the antibody determined. The more antigen there is in the testsample the less radioactive antigen will bind to the antibody. Acompetitive binding assay may also be used with non-radioactive antigen,using antigen or an analogue linked to a reporter molecule. The reportermolecule may be a fluorochrome, phosphor or laser dye with spectrallyisolated absorption or emission characteristics. Suitable fluorochromesinclude fluorescein, rhodamine, phycoerythrin and Texas Red. Suitablechromogenic dyes include diaminobenzidine.

Other reporters include macromolecular colloidal particles orparticulate material such as latex beads that are coloured, magnetic orparamagnetic, and biologically or chemically active agents that candirectly or indirectly cause detectable signals to be visually observed,electronically detected or otherwise recorded. These molecules may beenzymes which catalyse reactions that develop or change colours or causechanges in electrical properties, for example. They may be molecularlyexcitable, such that electronic transitions between energy states resultin characteristic spectral absorptions or emissions. They may includechemical entities used in conjunction with biosensors. Biotin/avidin orbiotin/streptavidin and alkaline phosphatase detection systems may beemployed.

The signals generated by individual antibody-reporter conjugates may beused to derive quantifiable absolute or relative data of the relevantantibody binding in samples (normal and test).

The present invention also provides the use of a specific binding memberas above for measuring antigen levels in a competition assay, that is tosay a method of measuring the level of antigen in a sample by employinga specific binding member as provided by the present invention in acompetition assay. This may be where the physical separation of boundfrom unbound antigen is not required. Linking a reporter molecule to thespecific binding member so that a physical or optical change occurs onbinding is one possibility. The reporter molecule may directly orindirectly generate detectable, and preferably measurable, signals. Thelinkage of reporter molecules may be directly or indirectly, covalently,e.g. via a peptide bond or non-covalently. Linkage via a peptide bondmay be as a result of recombinant expression of a gene fusion encodingantibody and reporter molecule.

The present invention also provides for measuring levels of antigendirectly, by employing a specific binding member according to theinvention for example in a biosensor system.

The mode of determining binding is not a feature of the presentinvention and those skilled in the art are able to choose a suitablemode according to their preference and general knowledge.

As noted, in various aspects and embodiments, the present inventionextends to a specific binding member which competes for binding to IL-13with any specific binding member defined herein, e.g. BAK502G9 IgG4.Competition between binding members may be assayed easily in vitro, forexample by tagging a specific reporter molecule to one binding memberwhich can be detected in the presence of other untagged bindingmember(s), to enable identification of specific binding members whichbind the same epitope or an overlapping epitope.

Competition may be determined for example using ELISA in which IL-13 isimmobilised to a plate and a first tagged binding member along with oneor more other untagged binding members is added to the plate. Presenceof an untagged binding member that competes with the tagged bindingmember is observed by a decrease in the signal emitted by the taggedbinding member.

In testing for competition a peptide fragment of the antigen may beemployed, especially a peptide including an epitope of interest. Apeptide having the epitope sequence plus one or more amino acids ateither end may be used. Such a peptide may be said to “consistessentially” of the specified sequence. Specific binding membersaccording to the present invention may be such that their binding forantigen is inhibited by a peptide with or including the sequence given.In testing for this, a peptide with either sequence plus one or moreamino acids may be used.

Specific binding members which bind a specific peptide may be isolatedfor example from a phage display library by panning with the peptide(s).

The present invention further provides an isolated nucleic acid encodinga specific binding member of the present invention. Nucleic acid mayinclude DNA and/or RNA. In a preferred aspect, the present inventionprovides a nucleic acid which codes for a CDR or set of CDR's or VHdomain or VL domain or antibody antigen-binding site or antibodymolecule, e.g. scFv or IgG4, of the invention as defined above.

The present invention also provides constructs in the form of plasmids,vectors, transcription or expression cassettes which comprise at leastone polynucleotide as above.

The present invention also provides a recombinant host cell whichcomprises one or more constructs as above. A nucleic acid encoding anyCDR or set of CDR's or VH domain or VL domain or antibodyantigen-binding site or antibody molecule, e.g. scFv or IgG4 asprovided, itself forms an aspect of the present invention, as does amethod of production of the encoded product, which method comprisesexpression from encoding nucleic acid therefor. Expression mayconveniently be achieved by culturing under appropriate conditionsrecombinant host cells containing the nucleic acid. Following productionby expression a VH or VL domain, or specific binding member may beisolated and/or purified using any suitable technique, then used asappropriate.

Specific binding members, VH and/or VL domains, and encoding nucleicacid molecules and vectors according to the present invention may beprovided isolated and/or purified, e.g. from their natural environment,in substantially pure or homogeneous form, or, in the case of nucleicacid, free or substantially free of nucleic acid or genes origin otherthan the sequence encoding a polypeptide with the required function.Nucleic acid according to the present invention may comprise DNA or RNAand may be wholly or partially synthetic. Reference to a nucleotidesequence as set out herein encompasses a DNA molecule with the specifiedsequence, and encompasses a RNA molecule with the specified sequence inwhich U is substituted for T, unless context requires otherwise.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, mammalian cells, plant cells, yeast and baculovirus systemsand transgenic plants and animals. Mammalian cell lines available in theart for expression of a heterologous polypeptide include Chinese hamsterovary (CHO) cells, HeLa cells, baby hamster kidney cells, NSO mousemelanoma cells, YB2/0 rat myeloma cells, human embryonic kidney cells,human embryonic retina cells and many others. A common, preferredbacterial host is E. coli.

The expression of antibodies and antibody fragments in prokaryotic cellssuch as E. coli is well established in the art. For a review, see forexample Plückthun, A. Bio/Technology 9: 545-551 (1991). Expression ineukaryotic cells in culture is also available to those skilled in theart as an option for production of a specific binding memberfor exampleChadd HE and Chamow SM (2001) 110 Current Opinion in Biotechnology 12:188-194, Andersen D C and Krummen L (2002) Current Opinion inBiotechnology 13: 117, Larrick J W and Thomas D W (2001) Current opinionin Biotechnology 12:411-418.

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorsequences, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. Vectors may be plasmids, viral e.g.‘phage, or phagemid, as appropriate. For further details see, forexample, Molecular Cloning: a Laboratory Manual: 3rd edition, Sambrookand Russell, 2001, Cold Spring Harbor Laboratory Press. Many knowntechniques and protocols for manipulation of nucleic acid, for examplein preparation of nucleic acid constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins, are described in detail in Current Protocols in MolecularBiology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1988,Short Protocols in Molecular Biology: A Compendium of Methods fromCurrent Protocols in Molecular Biology, Ausubel et al. eds., John Wiley& Sons, 4^(th) edition 1999. The disclosures of Sambrook et al. andAusubel et al. (both) are incorporated herein by reference.

Thus, a further aspect of the present invention provides a host cellcontaining nucleic acid as disclosed herein. Such a host cell may be invitro and may be in culture. Such a host cell may be in vivo. In vivopresence of the host cell may allow intracellular expression of thespecific binding members of the present invention as “intrabodies” orintracellular antibodies. Intrabodies may be used for gene therapy[112].

A still further aspect provides a method comprising introducing suchnucleic acid into a host cell. The introduction may employ any availabletechnique. For eukaryotic cells, suitable techniques may include calciumphosphate transfection, DEAE-Dextran, electroporation, liposome-mediatedtransfection and transduction using retrovirus or other virus, e.g.vaccinia or, for insect cells, baculovirus. Introducing nucleic acid inthe host cell, in particular a eukaryotic cell may use a viral or aplasmid based system. The plasmid system may be maintained episomally ormay incorporated into the host cell or into an artificial chromosome[110,111]. Incorporation may be either by random or targeted integrationof one or more copies at single or multiple loci. For bacterial cells,suitable techniques may include calcium chloride transformation,electroporation and transfection using bacteriophage.

The introduction may be followed by causing or allowing expression fromthe nucleic acid, e.g. by culturing host cells under conditions forexpression of the gene.

In one embodiment, the nucleic acid of the invention is integrated intothe genome (e.g. chromosome) of the host cell. Integration may bepromoted by inclusion of sequences which promote recombination with thegenome, in accordance with standard techniques.

The present invention also provides a method which comprises using aconstruct as stated above in an expression system in order to express aspecific binding member or polypeptide as above.

Aspects and embodiments of the present invention will now be illustratedby way of example with reference to the following experimentation.

EXAMPLE 1

Isolation of Anti-IL-13 scFv

ScFv Antibody Repertoire

A large single chain Fv (scFv) human antibody library derived fromspleen lymphocytes from 20 donors and cloned into a phagemid vector wasused for selections [66].

Selection of scFv

ScFv which recognised IL-13 were isolated from phage display librariesin a series of repeated selection cycles on recombinant bacteriallyderived human or murine IL-13 (Peprotech) essentially as described in[67]. In brief, following incubation with the library, the immobilisedantigen, which had been pre-coupled to paramagnetic beads, and boundphage were recovered by magnetic separation whilst unbound phage werewashed away. Bound phage was then rescued as described by Vaughan et al[67] and the selection process repeated. Different solid surfaces andcapture methods were used at different rounds of selection to reducenon-specific binding. Antigen was either covalently coupled to beads(Dynabeads M-270 carboxylic acid) or modified by biotinylation prior tosecondary capture by streptavidin-coated beads (Dynabeads M-280)according to manufacturer's protocols (Dynal). A representativeproportion of clones from the output of selection rounds were subjectedto DNA sequencing as described in Vaughan et al [67] and Osbourn et al[70]. Unique clones were assessed for their ability to neutralise IL-13as purified scFv preparations in IL-13 dependent cell proliferationassays.

Ribosome display libraries were created and screened for scFv thatspecifically recognised recombinant, bacterially derived human or murineIL-13 (Peprotech), essentially as described in Hanes et al [113].Initially the BAK278D6 lead clone from the initial selections wasconverted to ribosome display format, and this template was subsequentlyused for library creation. On the DNA level, a T7 promoter was added atthe 5′-end for efficient transcription to mRNA. On the mRNA level, theconstruct contained a prokaryotic ribosome-binding site (Shine-Dalgarnosequence). At the 3′ end of the single chain, the stop codon was removedand a portion of gIII (gene III) was added to act as a spacer [113].

Ribosome display libraries derived from BAK278D6 were created bymutagenesis of antibody complementarity determining regions (CDRs) wherePCR reactions were performed with non-proof reading Taq polymerase.Affinity-based selections were performed whereby, following incubationwith the library, the biotinylated human-IL-13 was captured bystreptavidin-coated paramagnetic beads (Dynal M280) and bound tertiarycomplexes (mRNA-ribosome-scFv-IL-13) were recovered by magneticseparation whilst unbound complexes were washed away. The mRNA encodingthe bound scFvs were then recovered by RT-PCR as described in Hanes etal [113] and the selection process repeated with decreasingconcentrations (100 nM-100 pM over 5 rounds) of biotinylated human IL-13present during the selection.

Error-prone PCR was also used to further increase library size. Threeintensities of error were employed (2.0, 3.5 and 7.2 mutations per 1,000bp after a standard PCR reaction, as described in manufacturer'sprotocol (Clontech)) during the selection regime. Initial error pronePCR reactions took place before round one selections commenced at 100nM. A subsequent round of error prone PCR was performed before roundthree selections at 10 nM biotinylated human-IL-13. As above, arepresentative proportion of clones from the output of selection roundswere subjected to DNA sequencing as described in Vaughan et al [67] andOsbourn et al [70]. Unique clones were assessed for their ability toneutralise IL-13 as purified scFv preparations in IL-13 dependent cellproliferation assays.

EXAMPLE 2

Neutralisation Potency of Anti-IL-13 scFv in the IL-13 Dependent TF-1Cell Proliferation Assay

The neutralisation potency of purified scFv preparations against humanand murine IL-13 bioactivity was assessed using TF-1 cell proliferationassay. Purified scFv preparations were prepared as described in Example3 of WO01/66754. Protein concentrations of purified scFv preparationswere determined using the BCA method (Pierce). TF-1 is a humanpremyeloid cell line established from a patient with erythroleukemia[68]. The TF-1 cell line is factor dependent for survival andproliferation. In this respect TF-1 cells responded to either human ormurine IL-13 [69] and were maintained in media containing human GM-CSF(4 ng/ml, R&D Systems). Inhibition of IL-13 dependent proliferation wasdetermined by measuring the reduction in incorporation of tritiatedthymidine into the newly synthesized DNA of dividing cells.

TF-1 Cell Assay Protocol

TF-1 cells were obtained from R&D Systems and maintained according tosupplied protocols. Assay media comprised RPMI-1640 with GLUTAMAX I(Invitrogen) containing 5% foetal bovine serum (JRH) and 1% sodiumpyruvate (Sigma). Prior to each assay, TF-1 cells were pelleted bycentrifugation at 300×g for 5 mins, the media removed by aspiration andthe cells resuspended in assay media. This process was repeated twicewith cells resuspended at a final concentration of 10⁵ cells/ml in assaymedia. Test solutions of antibody (in triplicate) were diluted to thedesired concentration in assay media. An irrelevant antibody notdirected at IL-13 was used as a negative control. Recombinantbacterially derived human or murine IL-13 (Peprotech) was added to afinal concentration of 50 ng/ml when mixed with the appropriate testantibody in a total volume of 100 μl/well in a 96 well assay plate. Theconcentration of IL-13 used in the assay was selected as the dose thatat final assay concentration gave approximately 80% of the maximalproliferative response. All samples were incubated for 30 minutes atroom temperature. 100 μl of resuspended cells were then added to eachassay point to give a total assay volume of 200 μl/well. Assay plateswere incubated for 72 hours at 37° C. under 5% CO₂. 25 μl of tritiatedthymidine (10 μCi/ml, NEN) was then added to each assay point and assayplates were returned to the incubator for a further 4 hours. Cells wereharvested on glass fibre filter plates (Perkin Elmer) using a cellharvester. Thymidine incorporation was determined using a PackardTopCount microplate liquid scintillation counter. Data were analysedusing Graphpad Prism software.

Results

Despite alternating selection cycles between human and murine antigen nocross-reactive neutralising antibodies were obtained. Two distinctanti-human and one anti-murine IL-13 neutralising scFvs were obtainedfrom selections. BAK278D6 (VH SEQ ID NO: 13; VL SEQ ID NO: 14) andBAK167A11 (VH SEQ ID NO: 23; VL SEQ ID NO: 24) recognised human IL-13whilst BAK209B11 (VH SEQ ID NO: 25; VL SEQ ID NO: 26) recognised murineIL-13. BAK278D6 (FIG. 2) and BAK167A11 (FIG. 1) as scFv neutralised 25ng/ml human IL-13 with an IC₅₀ of 44 nM and 111 nM respectively.BAK209B11 (FIG. 3) as a scFv neutralised 25 ng/ml murine IL-13 with anIC₅₀ of 185 nM.

EXAMPLE 3

Neutralisation Potency of Lead Clones From Targeted Optimisation ofHeavy Chain CDR3 of Parental Clones in the IL-13 Dependent TF-1 CellProliferation Assay

Osbourn et al. [70] have demonstrated that targeted mutagenesis ofresidues within heavy chain CDR3 can significantly improve the affinityof antibodies. Selections were performed as described in Example 1, onscFv repertoires in which residues within the heavy chain CDR3 ofBAK278D6 (SEQ ID NO: 6) BAK167A11 (SEQ ID NO: 57) had been randomised bymutagenesis. Unique clones from the selection output were identified byDNA sequencing and their neutralising potency assessed as scFv in theTF-1 cell proliferation assay, as described in Example 2.

Results

Significant gains in potency were achieved for both lineages. The mostpotent clones from the BAK167A11 lineage were BAK615E3, BAK612B5 andBAK582F7 which as scFv had IC₅₀ of 3 nM (FIG. 1), 6.6 nM, 6.65 nMrespectively against 25 ng/ml human IL-13 in TF-1 cell proliferationassay. From the BAK278D6 lineage, the most potent clone was BAK502G9,which as scFv had IC₅₀ of 8 nM against 25 ng/ml human IL-13 in the TF-1cell proliferation assay (FIG. 2).

EXAMPLE 4

Neutralisation Potency of BAK167A11 and BAK278D6 Lineages AgainstNon-Human Primate IL-13 and an IL-13 Variant Associated with Asthma inthe TF-1 Factor Dependent Cell Proliferation Assay

Neither of the BAK167A11 and BAK278D6 human IL-13 neutralising lineageswere murine cross-reactive. The inventors therefore decided on thefollowing criteria for the lineage selected for further optimisation andclinical development: should preferably be cross-reactive with non-humanprimate IL-13 and should recognise a variant of IL-13, in which arginineat amino acid at position 130 is substituted for by glutamine (Q130R).This variant has been genetically associated with asthma and otherallergic diseases [37, 39, 41, 71]. Cross-reactivity was determined bythe ability of purified scFv preparations to bind non-human primateIL-13 and IL-13 variant by surface plasmon resonance (BIAcore) analysis.Functional activity was determined using the TF-1 cell proliferationassay.

Production of wild-type, variant and non-human primate IL-13 A cDNA forwild-type human IL-13 was obtained from InvivoGen and modified bysite-directed mutagenesis (Stratagene Quikchange® kit) to yield a cDNAencoding variant IL-13. The coding sequence for both rhesus andcynomolgus monkey IL-13 was obtained by PCR on genomic DNA templateusing degenerate primers based on the human IL-13 sequence. Bothnon-human primate (rhesus and cynomolgus) sequences were identical toeach other but differed from human IL-13 by seven amino acids (FIG. 19).Recombinant wild type, variant and non-human primate IL-13 weresubsequently expressed using the baculovirus expression system(Invitrogen). Expression constructs added a carboxyl terminus affinitytag to the expressed protein that allowed purification from insect cellconditioned media to near homogeneity.

Qualitative Binding Assay using BIAcore

The binding affinity of purified scFv preparations to non-human primate,variant and wild type IL-13 was determined by surface plasmon resonancemeasurements using a BIAcore 2000 Biosensor (BIAcore AB) as described inKarlsson et al [72]. In brief, IL-13 was coupled to CM5 sensorchipsusing an amine coupling kit (BIAcore) at a surface density ofapproximately 200 Ru and three concentrations of test scFv(approximately 350 nM, 175 nM and 88 nM) in HBS-EP buffer passed overthe sensor chip surface. The resulting sensorgrams were evaluated usingBIA evaluation 3.1 software to provide relative binding data.

TF-1 Assay Protocol

The assay was performed essentially as described in Example 2 with thefollowing modifications: non-human primate IL-13, human variant IL-13(Q130R) and wild type human IL-13 were used at concentrations of 50ng/ml, 25 ng/ml and 25 ng/ml respectively.

Results

BIAcore binding assay data suggested that BAK278D6 but not BAK167A11lineage had the required cross-reactivity profile for furthertherapeutic development (Table 2). This finding was supported bybioassay data demonstrating that BAK278D6 (FIG. 4) and BAK502G9 (FIG. 6)were able to neutralise human IL-13, the human IL-13 (Q130R) variant andnon-human primate IL-13 in the TF-1 cell proliferation assay with nearequivalent potency. In contrast, although BAK615E3 (VH SEQ ID NO: 33; VLSEQ ID NO: 34) had a significantly increased potency against human IL-13over its parent BAK167A11 (VH SEQ ID NO: 23; VL SEQ ID NO: 24) in theTF-1 cell proliferation assay (FIG. 1), neither clone bound non-humanprimate or variant IL-13 in the BIAcore binding assay.

Germlining Framework Regions of BAK278D6 and BAK502G9

The derived amino acid sequence of BAK278D6 VH (SEQ ID NO: 13) and VL(SEQ ID NO: 14) were aligned to the known human germline sequences inthe VBASE database [73] and the closest germline identified by sequencesimilarity. The closest germline for the VH domain of BAK278D6 (SEQ IDNO: 14) and its derivatives, was identified as DP14, a member of the VH1family. The BAK278D6 VH has 9 changes from the DP14 germline withinframework regions. The closest germline for the VL of BAK278D6 wasidentified as V_(λ)3 3 h. The BAK278D6 VL domain (SEQ ID NO: 14) hasonly 5 changes from the germline within framework regions. Frameworkregions of BAK278D6 and its derivatives were returned to germline bysite directed mutagenesis (Stratagene Quikchange kit) to identicallymatch native human antibodies.

EXAMPLE 5

Neutralisation Potency of Lead Clones From Targeted Optimisation ofHeavy Chain CDR1 and Heavy Chain CDR2 Sequences of BAK502G9 in the HumanIL-13 Dependent TF-1 Cell Proliferation Assay

A second phase of optimisation was performed using BAK502G9 sequence,with germlined framework regions, as a template. Selections wereperformed essentially as described in Example 1 on scFv repertories inwhich either residues within the heavy chain CDR1 or heavy chain CDR2 ofBAK502G9 had been randomised by mutagenesis. Unique clones from theselection output were identified by DNA sequencing and theirneutralising potency assessed as purified scFv preparations in the TF-1cell proliferation assay as described in Example 2. Vectors wereconstructed for the most potent scFv clones to allow re-expression aswhole human IgG4 antibody as described by Persic et al. (1997 Gene 187;9-18) with a few modifications. An oriP fragment was included in thevectors to facilitate use with HEK-EBNA 293 cells and to allow episomalreplication. The VH variable domain was cloned into the polylinkerbetween the secretion leader sequence and the human gamma 4 constantdomain of the expression vector pEU8.1(+). The VL variable domain wascloned into the polylinker between the secretion leader sequence and thehuman lambda constant domain of the expression vector pEU4.1(−).

Whole antibody was purified from conditioned media from EBNA-293 cellsco-transfected with constructs expressing heavy and light chains byprotein A affinity chromatography (Amersham Pharmacia). The purifiedantibody preparations were sterile filtered and stored at 4° C. inphosphate buffered saline (PBS) prior to evaluation. Proteinconcentration was determined by measuring absorbance at 280 nm using theBCA method (Pierce). Reformatted human IgG4 whole antibodies werecompared to commercially available anti-human IL-13 antibodies in theTF-1 proliferation assay described in Example 2.

Results

As demonstrated in FIG. 5, the commercial antibody B-B13, (mouse IgG1-Euroclone 5) was shown to be significantly more potent against humanIL-13 than the commercial antibody JES10-5A2 (rat IgG1-Biosource) withIC₅₀ of 1021 pM and 471 pM respectively. Eight clones, namely,BAK1111D10, BAK1166G02,

BAK1167F02, BAK1167F04, BAK1183H4, BAK1184C8, BAK1185E1, BAK1185F8,derived from BAK502G9 (and so “BAK502G9 lineage”), in which the heavychain CDR1 or CDR2 had been targeted, showed improved potency as scFvover the commercial antibodies. These improvements were maintained onconversion to whole antibody human IgG4. Each of these VH and VL domainsindividually and in the respective pairings of these claims representsan aspect or embodiment of the present invention, as do specific bindingmembers for IL-13 that comprise one or more of them, also specificbinding members comprising one or more CDR's from the BAK502G9 lineageclones, preferably a VH domain comprising a BAK502G9 lineage set ofHCDR's and/or a VL domain comprising a BAK502G9 lineage set of LCDR's.These may be employed in any and all aspects of the invention asdisclosed elsewhere herein. Derivatives of BAK502G9 as whole antibodies(IgG4) had an IC₅₀ ranging from 244 pM to 283 pM. BAK502G9 as a wholeantibody IgG4 had an IC₅₀ of 384 pM. In summary, major improvements inpotency could be obtained by targeting heavy chain CDR1 (SEQ ID NO:7) orCDR2 (SEQ ID NO: 8) of BAK502G9. Statistical comparisons to B-B13 weremade using an ANOVA followed by a Dunnett's post test analysis (InStatsoftware).

Further Characterisation

Selected anti-human antibodies from the BAK278D6 lineage underwentfurther characterisation to determine their specificity. These includedBAK502G9 (VH SEQ ID NO: 15; VL SEQ ID NO: 16) and its derivativesBAK1167F2 (VH SEQ ID NO: 35; VL SEQ ID NO: 36) and BAK1183H4 (VH SEQ IDNO: 37; VL SEQ ID NO:

38), which are representative examples of clones with modifications toheavy chain CDR1 and heavy chain CDR2 of BAK502G9 respectively.

EXAMPLE 6

Neutralisation Potency of Lead Clones From Targeted Optimisation ofHeavy Chain CDR1 and Heavy Chain CDR2 Sequences of BAK502G9 AgainstNon-Human Primate IL-13 and an IL-13 Variant Associated with Asthma inthe TF-1 Factor Dependent Cell Proliferation Assay

Cross-reactivity of anti-human IL-13 antibodies was determined by theirability to inhibit non-human primate IL-13 and IL-13 variant mediatedTF-1 cell proliferation as described in Example 4.

Results

Optimised anti-human IL-13 antibodies BAK1167F2 (VH SEQ ID NO: 35; VLSEQ ID NO: 36) and BAK1183H4 (VH SEQ ID NO: 37; VL SEQ ID NO: 38)maintained the specificity of their parent BAK502G9 (VH SEQ ID NO: 15;VL SEQ ID NO: 16) (FIG. 6). Potency gains against wild type IL-13 werereflected in their ability to neutralise non-human primate IL-13 and anIL-13 variant with substantially equivalent potency. The IC₅₀ forBAK502G9 against human, human variant and non-human primate IL-13 were1.4 nM, 1.9 nM and 2.0 nM respectively. The IC₅₀ for BAK1167F2 againsthuman, human variant and non-human primate IL-13 were 1.0 nM, 1.1 nM and1.3 nM respectively. The IC₅₀ for BAK1183H4 against human, human variantand non-human primate IL-13 were 0.9 nM, 1.0 nM and 1.6 nM respectively.These clones are suitable for therapeutic use.

EXAMPLE 7

Neutralising Potency of Lead Anti-Human IL-13 Antibodies Against NativeHuman IL-13 in HDLM-2 Cell Proliferation Assay

The human IL-13 sequence has four potential N-glycosylation sites. Theinventors have demonstrated the ability of BAK278D6 and its derivativesto neutralise recombinant IL-13 expressed either in bacterial orbaculovirus expression systems. Although, there is evidence that manyprocessing events known in mammalian systems do also occur in insectsthere are key differences in protein glycosylation, particularlyN-glycosylation [74].

The inventors investigated the ability of BAK278D6 derivatives toneutralise native IL-13 released from human cells.

HDLM-2 cells were isolated by Drexler et al [75] from a patient withHodgkin's disease. Skinnider et al [76] demonstrated that HDLM-2 cellproliferation was in part dependent on autocrine and paracrine releaseof IL-13. Lead anti-human IL-13 antibodies were assessed for theirability to inhibit HDLM-2 cell proliferation mediated by the release ofnative (or naturally occurring) IL-13.

HDLM-2 Cell Assay Protocol

HDLM-2 cells were obtained from the Deutsche Sammlung vonMikroorganismen and Zellkulturen (DSMZ) and maintained according tosupplied protocols. Assay media comprised RPI-1640 with Glutamax I(Invitrogen) containing 20% foetal bovine serum. Prior to each assay,the cells were pelleted by centrifugation at 300×g for 5 min, the mediaremoved by aspiration and the cells resuspended in fresh media. Thisprocess was repeated three times and the cells were finally resuspendedto a final concentration of 2×10⁵ cells/ml in assay media. 50 μl ofresuspended cells were added to each assay point in a 96 well assayplate. Test solutions of antibodies (in triplicate) were diluted to thedesired concentration in assay media. An irrelevant isotype antibody notdirected at IL-13 was used as a negative control. The appropriate testantibody in a total volume of 50 μl/well were added to the cells, eachassay point giving a total assay volume of 100 μl/well. Assay plateswere incubated for 72 hours at 37° C. under 5% CO₂. 25 μl of tritiatedthymidine (10 μCi/ml, NEN) was then added to each assay point and assayplates were returned to the incubator for a further 4 hours. Cells wereharvested on glass fibre filter plates (Perkin Elmer) using a cellharvester. Thymidine incorporation was determined using a PackardTopCount microplate liquid scintillation counter. Data were analysedusing Graphpad Prism software.

Results

As demonstrated in FIG. 7, BAK502G9 (VH SEQ ID NO: 15; VL SEQ ID NO:16), and its derivatives BAK1183H4 (VH SEQ ID NO: 37; VL SEQ ID NO: 38)and BAK1167F2 (VH SEQ ID NO: 35; VL SEQ ID NO: 36) were able to cause adose dependent inhibition of cell proliferation with relative potenciessimilar to those observed in other bioassays. IC₅₀ for BAK502G9,BAK1183H4, BAK1167F2 as human IgG4 were 4.6 nM, 3.5 nM and 1.1 nMrespectively. IC₅₀ for the commercial antibodies JES10-5A2 and B-B13were 10.7 nM and 16.7 nM respectively.

EXAMPLE 8

Neutralising potency of lead anti-human IL-13 antibodies against IL-13dependent responses in disease relevant primary cells

Secondary bioassays were performed using primary cells and readouts morerelevant to airway disease. These included eotaxin release from normalhuman lung fibroblasts (NHLF) and vascular adhesion molecule 1 (VCAM-1)upregulation on the surface of human umbilical vein endothelial cells(HUVEC). Both IL-13 dependent responses could contribute to eosinophilrecruitment, a feature of the asthma phenotype [92].

NHLF Assay Protocol

IL-13 has been shown to cause eotaxin release from lung fibroblasts[77][78] [79]. Factor dependent eotaxin release from NHLF was determined byELISA.

NHLF were obtained from Biowhittaker and maintained according tosupplied protocols. Assay media was FGM-2 (Biowhittaker).

Test solutions of antibody (in triplicate) were diluted to the desiredconcentration in assay media. An irrelevant antibody not directed atIL-13 was used as a negative control. Recombinant bacterially-derivedhuman IL-13 (Peprotech) was subsequently added to a final concentrationof 10 ng/ml when mixed with the appropriate test antibody in a totalvolume of 200 μl. The concentration of IL-13 used in the assay wasselected as the dose that gave an approximately 80% of the maximalresponse. All samples were incubated for 30 minutes at room temperature.Assay samples were then added to NHLF that had been preseeded at adensity of 1×10⁴ cells per well in 96-well assay plates. Assay plateswere incubated at 37° C. for 16-24 hours at 37° C. under 5% CO₂. Assayplates were centrifuged at 300 ×g for 5 minutes to pellet detachedcells. Eotaxin levels in the supernatant were determined by ELISA usingreagents and methods described by the manufacturer (R&D Systems). Datawere analysed using Graphpad Prism software.

Results

BAK278D6 lineage clones were able to inhibit human IL-13 dependenteotaxin release from NHLF. Relative potency was similar to that observedin the TF-1 cell proliferation assay (FIG. 8). BAK502G9 (VH SEQ ID NO:15; VL SEQ ID NO: 16), BAK1183H4 (VH SEQ ID NO: 37; VL SEQ ID NO: 38),BAK1167F2 (VH SEQ ID NO: 35; VL SEQ ID NO: 36) had IC₅₀ of 207 pM, 118pM and 69 pM respectively against 10 ng/ml human IL-13. Commercialantibodies JES10-5A2 and B-B13 had IC₅₀ of 623 pM and 219 pMrespectively.

HUVEC Assay Protocol

IL-13 has been shown to upregulate expression of VCAM-1 on cell surfaceof HUVECs [80, 81]. Factor dependent VCAM-1 expression was determined bydetection of upregulation of VCAM-1 receptor cellular expression using atime-resolved fluorescence read out.

HUVEC were obtained from Biowhittaker and maintained according tosupplied protocols. Assay media was EGM-2 (Biowhittaker). Test solutionsof antibody (in triplicate) were diluted to the desired concentration inassay media. An irrelevant antibody not directed at IL-13 was used as anegative control. Recombinant bacterially derived human IL-13(Peprotech) was added to a final concentration of 10 ng/ml when mixedwith the appropriate test antibody in a total volume of 200 μl. Theconcentration of IL-13 used in the assay was selected as the dose thatgave approximately 80% of the maximal response. All samples wereincubated for 30 minutes at room temperature. Assay samples were thenadded to HUVEC that had been preseeded at 4×10⁴ cells per well in96-well assay plates. Assay plates were incubated at 37° C. for 16-20hours under 5% CO₂. Assay media was then removed by aspiration andreplaced with blocking solution (PBS containing 4% dried Marvel® milkpowder). Assay plates were incubated at room temperature for 1 hour atroom temperature. Wells were washed three times with PBST Tween before100 μl (1:500 dilution in PBST/1% Marvel®) of biotinylated anti-VCAM-1antibody (Serotec) was added to each well. Assay plates were incubatedat room temperature for 1 hour. Wells were washed three times withDelfia wash buffer (Perkin Elmer) before 100 μl of Europium-labelledStreptavidin or anti-murine IgG1 (1:1000 dilution in Delfia assaybuffer, Perkin Elmer) was added to each well. Assay plates were thenincubated at RT for 1 hour. Wells were washed 7 times with Delfia washbuffer (Perkin Elmer). Finally, 100 μl of enhancement solution (PerkinElmer) was added to each well and fluorescence intensity was determinedusing the Wallac 1420 VICTOR2 plate reader (Standard Europium protocol).Data were analysed using Graphpad Prism software.

Results

Typical data for BAK502G9 (VH SEQ ID NO: 15; VL SEQ ID NO: 16),BAK1183H4 (VH SEQ ID NO: 37; VL SEQ ID NO: 38), BAK1167F2 (VH SEQ ID NO:35; VL SEQ ID NO: 36) as whole antibody human IgG4 are shown in FIG. 9.Relative potency was similar to the observed in the TF-1 cellproliferation assay. IC₅₀ for BAK502G9, BAK1183H4 and BAK1167F2 were 235pM, 58 pM and 55 pM respectively against lOng/ml human IL-13.

EXAMPLE 9

Neutralisation Potency of anti-IL-13 Antibodies Against IL-1/3 and IL-4Dependent VCAM-1 Upregulation

The specificity of the BAK278D6 lineage of clones was assessed in amodification of the HUVEC bioassay. Together with IL-13, both IL-4 andIL-1β have been shown to upregulate expression of VCAM-1 on cell surfaceof HUVECs [80, 81].

HUVEC Assay Protocol

The assay was performed essentially as described in Example 5 with thefollowing modifications. Recombinant human IL-1β and IL-4 (R&D Systems)were used in place of human IL-13 at 0.5 ng/ml and 1 ng/ml respectivelyand represented the dose that gave approximately 80% of the maximalresponse.

Results

None of the clones evaluated from the BAK278D6 lineage neutralisedVCAM-1 upregulation in response to either human IL-1β or IL-4 and thusdemonstrated specificity for IL-13 (FIG. 10). IL-4 is most closelyrelated to IL-13, sharing 30% sequence identity at the amino acid level[82].

EXAMPLE 10

Neutralisation Potency of BAK209B11 as a Human IgG4 in a Murine IL-13Dependent Murine B9 Cell Proliferation Assay

BAK209B11, identified as an anti-murine IL-13 neutralising clone as ascFv as described in Example 1, was reformatted as a whole antibodyhuman IgG4 as described in Example 5 and its potency evaluated in themurine IL-13 dependent B9 cell proliferation assay. B9 is a murineB-cell hybridoma cell line [83]. B9 is factor dependent for survival andproliferation. In this respect B cells respond to murine IL-13 and aremaintained in media containing human IL-6 (50 pg/ml, R&D Systems).Inhibition of murine IL-13 dependent proliferation was determined bymeasuring the reduction in incorporation of tritiated thymidine into thenewly synthesized DNA of dividing cells.

B9 cell Assay Protocol

B9 cells were obtained from European Collection of Animal Cell CultureECACC and maintained according to supplied protocols. The assay wasperformed essentially as described for the TF-1 assay in Example 2 butwith the following modifications. Assay media comprised RPMI-1640 withGLUTAMAX I (Invitrogen) containing 5% foetal bovine serum (Hyclone) and50 μM 2-mercaptoethanol (Invitrogen). Recombinant bacterially derivedmurine IL-13 (Peprotech) replaced human IL-13 with a final assayconcentration of 1 ng/ml.

Results

BAK209B11 (VH SEQ ID NO: 25; VL SEQ ID NO: 26) as a human IgG4neutralised 1 ng/ml murine IL-13 with an IC₅₀ of 776 pM in the B9 assay(FIG. 11). BAK209B11 therefore represents a useful tool to investigatethe role of IL-13 in murine models of disease. This is clearlydemonstrated in Example 12, which demonstrates the efficacy of BAK209B11in a murine model of acute pulmonary inflammation.

EXAMPLE 11

Affinity Determination of Anti-IL-13 Antibodies by BIAcore Analysis

The affinity of BAK502G9 (VH SEQ ID NO: 15; VL SEQ ID NO: 16), BAK1167F2(VH SEQ ID NO: 35; VL SEQ ID NO: 36) and BAK1183H4 (VH SEQ ID NO: 37; VLSEQ ID NO: 38) for human IL-13 and BAK209B11 (VH SEQ ID NO: 25; VL SEQID NO: 26) for murine IL-13 as human IgG4 were determined by surfaceplasmon resonance measurements using a BIAcore 2000 Biosensor (BIAcoreAB) essentially as described in [72]. In brief, antibodies were coupledto CM5 sensorchips using an amine coupling kit (BIAcore) at a surfacedensity of approximately 500 Ru and a serial dilution of IL-13 (between50 nM to 0.78 nM) in HBS-EP buffer was passed over the sensorchipsurface. The resulting sensorgrams were evaluated using BIA evaluation3.1 software to provide kinetic data.

Results

BAK502G9, BAK1167F2 and BAK1183H4 IgG4 bound human IL-13 with highaffinity with Kd of 178 pM, 136 pM and 81 pM respectively correspondingto their relative potency in cell based assays. BAK209B11 bound murineIL-13 with affinity of 5.1 nM (Table 3) .

EXAMPLE 12 Efficacy of BAK209B11 in a Murine Model of Acute AllergicPulmonary Inflammation

Murine model of acute allergic pulmonary inflammation

The effect of BAK209B11 (VH SEQ ID NO: 25; VL SEQ ID NO: 26), ananti-murine IL-13 neutralising human IgG4 antibody, was investigated ina murine of acute allergic pulmonary inflammation. This model wasperformed essentially as described by Riffo-Vasquez et al [84] and ischaracterised at its endpoint by increased bronchial alveolar lavage(BAL) IL-13 (FIG. 12), cellular infiltration into the lung and BAL (FIG.13), increased serum IgE levels and airways hyperresponsiveness (AHR).

Model protocol

Female Balb/C mice (Charles River UK) were treated with eitheranti-murine IL-13 antibody BAK209B11 (at 12, 36, 119 or 357 μg doses) oran isotype matched control antibody (357 μg dose). On days 0 and 7, micein each group were sensitised by intraperitoneal injection of 10 μg ofovalbumin (Ova) in 0.2 ml of the vehicle (saline containing 2% A1₂0₃(Rehydragel) as an adjuvant). A separate control group of non-sensitisedmice received an equal volume of the vehicle. Mice were challenged withovalbumin on days 14, 15 and 16. Ovalbumin was diluted to 1% (w/v) insterile saline prior to nebulisation. All inhalation challenges wereadministered in a Plexiglas exposure chamber. Ova was aerosolised usinga deVilbiss Ultraneb 2000 nebuliser (Sunrise Medical) in a series ofthree exposures of 20 minutes separated by 1 hour intervals.

BAK209B11 or an irrelevant human IgG4 were administered intravenously, 1day prior to first challenge and then 2 hours prior to each subsequentchallenge (4 doses in total). The model ended at day 17, 24 hours postfinal challenge. Blood (serum) and BAL were collected. Serum was assayedfor total IgE. BAL was obtained by injecting 3 aliquots of saline (0.3ml, 0.3 ml and 0.4 ml) and pooling samples. Total leukocytes anddifferential cell counts were obtained from BAL cells.

Results

Ovalbumin challenge of sensitised mice caused a significant (p<0.05)increase in total BAL cell recruitment over non-sensitised butchallenged animals. This recruitment was dose-dependently inhibited byBAK209B11; significant (p<0.05) inhibition was seen with 36 μgBAK209B11, but not control antibody (FIG. 13). Similar effects were alsoseen on eosinophils (FIG. 14) and neutrophils (FIG. 15) with significant(p<0.05) inhibition of cellular influx at a minimum BAK209B11 dose of 36μg. This inhibition was not seen with the control antibody. Lymphocyteswere also induced in sensitised but not non-sensitised mice uponchallenge. This induction was dose-dependently inhibited by BAK209B11,with maximal inhibition seen with 36 μg BAK209B11. Control antibody hadno effect (FIG. 16). Although monocyte/macrophages were not induced insensitised animals when compared to non-sensitised animals, backgroundlevels were depressed by 36 μg BAK209B11, but not by control antibody(FIG. 17). Serum IgE levels were significantly increased in sensitisedanimals when compared to non-sensitised after challenge (p<0.05). Thisincrease was decreased after treatment with 36 μg BAK209B11 but not bythe control antibody.

In summary, systemic administration of BAK209B11, a murine IL-13neutralising antibody, but not control antibody inhibited inflammatorycell influx and the upregulation of serum IgE levels caused bysensitisation and subsequent challenge with ovalbumin in a murine modelof allergic inflammation.

Examples 13 to 20 are prophetic.

EXAMPLE 13

Efficacy of BAK209B11 in the Lloyd Murine Model of Acute PulmonaryInflammation

Murine model of acute allergic pulmonary inflammation

The effect of BAK209B11 (VH SEQ ID NO: 25; VL SEQ ID NO: 26), an antimurine IL-13 neutralising antibody, was investigated in a second murinemodel of acute allergic pulmonary inflammation. This model was performedessentially as described by McMillan et al. [85] and is characterised atits endpoint by increased BAL and lung tissue IL-13, cellularinfiltration into the lung and BAL, increased serum IgE levels andairways hyperresponsiveness (AHR).

Model Protocol

Female Balb/C mice (Charles River UK) were administered with variousdoses of anti-murine IL-13 antibody BAK209B11 or an isotype matchedcontrol antibody, as follows. On days 0 and 12, mice in each group weresensitised (SN) by intraperitoneal injection of 10 μg of ovalbumin (Ova)in 0.2 ml of the vehicle (saline containing 2 mg Al(OH)₃ as an adjuvant[calculated as described in Example 12]). A separate control group ofnon-sensitised mice (NS) received an equal volume of the vehicle. Micewere challenged with ovalbumin for 20 minutes on days 19, 20, 21, 22, 23and 24. Ovalbumin was diluted to 5% (w/v) in saline prior tonebulisation. All inhalation challenges were administered in a Plexiglasexposure chamber. Ova was aerosolised using a deVilbiss Ultraneb 2000nebuliser (Sunrise Medical). On days 18,19,20,21,22,23 and 24 mice wereadministered with various intraperitoneal doses (237 μg, 23.7 μg or 2.37μg; denoted in FIG. 21 by H, M and L) of anti-murine IL-13 antibodyBAK209B11 muIgG1 or an isotype matched control antibody (237 μg). Airwayfunction was assessed on days 0 and 25 by increasing methacholinechallenges and monitored using conscious plethysmography (Buxco). PC₅₀(concentration of methacholine required to increase baseline PenH by50%) was estimated for individual mice at both day 0 and day 25 from 4parameter unfixed curve fitting of methacholine dose-response curves.

The model ended at day 25, 24 hours post final challenge. Blood, serum,BAL and lung tissue were collected.

Results

Lung function was evaluated for individual animals at day 0(pre-treatment) and at day 25 (post-challenge) and was quantitated bycalculating PC₅₀ values (concentration of methacholine required toincrease baseline PenH by 50%) (FIG. 21A). An individuals airwayshyperresponsiveness (AHR) was determined by the change in log PC₅₀ atday 25 versus day 0 (log day 25 PC₅₀-log day 0 PC₅₀). This delta logPC₅₀was the primary endpoint of the study; PC₅₀ data log-transformed becauseof requirements of endpoint ANOVA. Individual changes were averagedwithin groups to generate group average delta log PC₅₀ (as shown in FIG.21B).

Ovalbumin challenge of sensitised mice caused a significant AHR comparedto non-sensitised and challenged mice (p<0.01). BAK209B11 caused a clearand dose-dependent decrease in AHR whereas the control antibody had noeffect.

EXAMPLE 14

Efficacy of BAK209B11 in the Gerard Murine Model of Acute PulmonaryInflammation

Murine model of acute allergic pulmonary inflammation

The effect of BAK209B11 (VH SEQ ID NO: 25; VL SEQ ID NO: 26), ananti-murine IL-13 neutralising human IgG4 antibody, was investigated ina third murine model of acute allergic pulmonary inflammation. Thismodel was performed essentially as described by Humbles et al. [86] andis characterised at its endpoint by increased BAL and lung tissue IL-13,cellular infiltration into the lung and BAL, increased serum IgE levelsand airways hyperresponsiveness (AHR).

Model Protocol

Female Balb/C mice (Charles River UK) were administered with variousdoses of anti-murine IL-13 antibody BAK209B11 or an isotype matchedcontrol antibody. On days 0, 7 and 14, mice in each group weresensitised (SN) by intraperitoneal injection of 10 μg of ovalbumin (Ova)in 0.2 ml of the vehicle (saline containing 1.125 mg Al(OH)₃ as anadjuvant [calculated as described in Example 12]). A separate controlgroup of non-sensitised mice (NS) received an equal volume of thevehicle. Mice were challenged with ovalbumin for 20 minutes on days 21,22, 23 and 24. Ovalbumin was diluted to 5% (w/v) in saline prior tonebulisation. All inhalation challenges were administered in a Plexiglasexposure chamber. Ova was aerosolised using a deVilbiss Ultraneb 2000nebuliser (Sunrise Medical).

The model ended at day 25, 24 hours post challenge. Blood, serum, BALand lung tissue were collected.

EXAMPLE 15

Efficacy of BAK209B11 in the Lloyd Chronic Model of PulmonaryInflammation

Murine model of chronic allergic pulmonary inflammation

The effect of BAK209B11 (VH SEQ ID NO: 25; VL SEQ ID NO: 26), an antimurine IL-13 neutralising human IgG4 antibody, was investigated in amodel of chronic allergic pulmonary inflammation. This model wasperformed essentially as described by Temelkovski et al. [87] and ischaracterised at its endpoint by cellular infiltration into the lung andBAL, increased serum IgE levels and airways hyperresponsiveness (AHR).

Model Protocol

Female Balb/C mice (Charles River UK) were dosed with various doses ofanti-murine IL-13 antibody BAK209B11 or an isotype matched controlantibody. On days 0 and 11, mice in each group were sensitised (SN) byintraperitoneal injection of 10 μg of ovalbumin (Ova) in 0.2 ml of thevehicle (saline containing 2 mg Al(OH)₃ as an adjuvant [calculated asdescribed in Example 12]). A separate control group of non-sensitisedmice (NS) received an equal volume of the vehicle. Mice were challengedwith ovalbumin for 20 minutes on days 18, 19, 20, 21, 22, 23, 28, 30,32, 35, 37, 39, 42, 44, 46, 49 and 51. Ovalbumin was diluted to 5% (w/v)in saline prior to nebulisation. All inhalation challenges wereadministered in a Plexiglas exposure chamber. Ova was aerosolised usinga deVilbiss Ultraneb 2000 nebuliser (Sunrise Medical).

The model ended at day 52, 24 hours post challenge. Blood, serum, BALand lung tissue were collected.

EXAMPLE 16

Efficacy of Anti-Human IL-13 Antibodies Against Exogenous Human IL-13Administered to the Murine Air Pouch Model

The effect of anti-human IL-13 antibodies on the pro-inflammatory actionof human IL-13 was investigated in a basic murine model. This model wasperformed essentially as described by Edwards et al [93] and wascharacterised at its endpoint by cellular infiltration into theairpouch.

Model Protocol

An air pouch was created on the back of female Balb/C mice bysubcutaneous injection of 2.5 mL of sterile air at day 0. The air pouchwas reinflated with another 2.5 mL sterile air at day 3. 2 μg huIL-13 in0.75% CMC was injected directly into the pouch at day 6. 24 hours laterthe mice were killed and the air pouch lavaged with 1mL heparinisedsaline. Antibody treatments were either given with the huIL-13 (into thepouch) or given systemically.

Results

Human IL-13, injected into the airpouch (i.po.), caused a significantlyincreased infiltration of total leukocytes (p<0.01) and eosinophils(p<0.01) at 24 hours post-challenge versus vehicle (0.75% carboxymethylcellulose (CMC) in saline i.po.) treated mice.

Locally administered BAK502G9 (200 mg, 20 mg or 2 mg intrapouch)significantly and dose-dependently inhibited the total leukocyte(p<0.01) and eosinophil (p<0.01) infiltration into the air pouch causedby 2 μg huIL-13 in 0.75% CMC.

Systemically administered BAK209B11 (30 mg/kg, 10 mg/kg and 1 mg/kg)also signficantly and dose-dependently inhibited the total leukocyte(p<0.01) and eosinophil (p<0.01) infiltration into the air pouch causedby 2 μg huIL-13 in 0.75% CMC.

EXAMPLE 17

Generation of Human IL-13 Knock-In/Murine IL-13 Knock Out TransgenicMice for the Purposes of Evaluating the Efficacy of Anti-Human IL-13Antibodies in Models of Pulmonary Allergic Inflammation

The present inventors have generated mice which express human, ratherthan murine IL-13 by gene targeting. The mouse IL-13 gene has beenreplaced from start to stop codon with the relevant portion of the humanIL-13 gene. This mouse strain expresses human IL-13, rather than mouseIL-13, in response to the same stimuli as in the wild-type mouse, as theendogenous IL-13 promoter and IL-13 pA tail remaining unchanged. It hasbeen shown that human IL-13 can bind to and signal through mouse IL-13receptors to generate the same physiological consequences as signallingcaused by mouse IL-13 ligating mouse IL-13 receptors. For exampleexogenous human IL-13 caused inflammatory cell recruitment into themurine air pouch (FIG. 18). These transgenic animals allow us toevaluate non-murine cross reactive anti-human IL-13 antibodies inestablished murine models of disease.

This mouse has been used in the acute allergic airway inflammationmodels (as described in examples 18 and 19) and chronic allergic airwayinflammation models (as described in Example 20) allowing the evaluationof anti-human IL-13 antibody pharmacology in allergic airway disease.

EXAMPLE 18

Efficacy of anti-human IL-13 antibodies in the hulL-13-transgenic Lloydmurine model of acute pulmonary inflammation

Murine model of acute allergic pulmonary inflammation

The effect of anti human IL-13 neutralising human IgG4 antibodies wereinvestigated in a murine model of acute allergic pulmonary inflammationusing the transgenic mice generated in example 17. This model wasperformed essentially as described by McMillan et al. [85] and example13. The model was characterised at its endpoint by increased BAL andlung tissue IL-13, cellular infiltration into the lung and BAL,increased serum IgE levels and airways hyperresponsiveness (AHR).

Model Protocol

The protocol for this model was as described in Example 13 except thatanti-human IL-13 antibodies were dosed instead of BAK209B11.

EXAMPLE 19

Efficacy of anti-human IL-13 antibodies in the hulL-13-transgenic Gerardmurine model of acute pulmonary inflammation

Murine Model of Acute Allergic Pulmonary Inflammation

The effect of anti human IL-13 neutralising human IgG4 antibodies wereinvestigated in another murine model of acute allergic pulmonaryinflammation using the transgenic mice generated in example 17. Thismodel was performed essentially as described by Humbles et al, [86] andin example 14. The model is characterised at its endpoint by increasedBAL and lung tissue IL-13, cellular infiltration into the lung and BAL,increased serum IgE levels and airways hyperresponsiveness (AHR).

Model Protocol

The protocol for this model was as described in Example 14 except thatanti-human IL-13 antibodies were dosed instead of BAK209B11.

EXAMPLE 20

Efficacy of Anti-Human IL-13 Antibodies in the hulL-13-Transgenic LloydChronic Model of Pulmonary Inflammation

The effect of anti human IL-13 neutralising human IgG4 antibodies wereinvestigated in a model of chronic allergic pulmonary inflammation usingthe transgenic mice generated in example 17. This model was performedessentially as described by Temelkovski et al. [87] and in Example 15and is characterised at its endpoint by cellular infiltration into thelung and BAL, increased serum IgE levels and airways hyperresponsiveness(AHR).

Model Protocol

The protocol for this model was as described in Example 15 except thatanti-human IL-13 antibodies were dosed instead of BAK209B11

EXAMPLE 21

Pharmacokinetics and Pharmacodynamics of Anti-Human IL-13 Antibodies inAscaris.suum-Allergic Cynomolgus Monkeys

The pharmacokinetics and pharmacodynamics of 502G9 were evaluated in 4allergic but non-challenged cynomolgus primates (2 male/2 female) aftera single 10 mg/kg i.v bolus dose. The experiment ran for 29 days. Theantibody's pharmacokinetic parameters were determined from a geomeanaverage serum-drug concentration curve and are detailed below in Table4.

In the same study serum IgE concentrations were also followed using ahuman IgE ELISA kit (Bethyl laboratories, USA).

Results

Serum IgE concentrations were significantly reduced after a single 10mg/kg i.v bolus dose of BAK502G9, from 100% control levels (predose) to66±10% of control values (p<0.05), at 4 and 5 days after dosing. Thislowering of serum IgE concentration recovered to 88±8% of control levelsby day 22 (see FIG. 20). Again these data were derived by normalisingeach animals serum IgE concentration to predose levels, where predoseconcentrations was 100%, and then averaging the curves from the 4animals tested.

The two male monkeys had relatively low predose total serum IgE (60ng/mL and 67 ng/mL). These IgE levels did not change in a fashionsuggesting a trend following treatment with BAK502G9 (FIG. 20B). The twofemale monkeys had relatively high predose total serum IgE (1209 ng/mLand 449 ng/mL). These IgE levels were decreased following treatment withBAK502G9, maximally by 60% at 7 days, and returning to approximatelypredose levels by 28 days post-administration (FIG. 20B).

These data provide indication that BAK502G9 lowers serum IgEconcentrations in animals with relatively high circulating IgE of IgE.

EXAMPLE 22

Efficacy of Anti-Human IL-13 Antibodies in Cynomolgus Models of DermalAllergy

The effects of anti-human IL-13 neutralising human IgG4 antibodies wereinvestigated in a primate model of acute allergic dermal inflammation.This model was performed by injecting human IL-13 and A.suum antigenintradermally into cynomolgus monkeys. 24-96h later, dermal biopsies andserum samples were taken. The model was characterised at its endpoint bycellular infiltration into the skin.

EXAMPLE 23

Efficacy of Anti-Human IL-13 Antibodies in Cynomolgus Models ofPulmonary Allergy

The effect of anti human IL-13 neutralising human IgG4 antibodies wereinvestigated in a primate model of acute allergic pulmonaryinflammation. This model was performed by exposing a.suum-allergiccynomolgus primates to nebulised a.suum antigen, thereby generating anallergic reaction. This allergy was characterized at its end point bycellular infiltration into the lung and BAL, increased serum IgE levelsand airways hyper-responsiveness.

Pharmacodynamics were additionally evaluated ex vivo using a flowcytometric method. CD23 is the high affinity IgE receptor and can beexpressed on peripheral human blood mononuclear cells. CD23 expressioncan be induced, in terms of the number of cells expressing CD23 and alsoin how much CD23 each cell expresses by both IL-13 and IL-4. The IL-13,but not IL-4, mediated response can be inhibited by anti-human IL-13antibodies.

Animals were preselected for entry into this 2-phase study on the basisof previously established AHR following nebulised antigen (ascaris suumextract) challenge. In phase I airway function was assessed duringintravenous histamine challenge on days 1 and 11. PC₃₀, the histaminedose required to generate a 30% increase in lung resistance (R_(L))above baseline, was determined from each histamine dose-response curve.On days 9 and 10, animals were challenged with individually tailoreddoses of nebulised antigen previously shown to generate a 40% increasein R_(L) as well as a 35% decrease in dynamic compliance (C_(DYN)).Historically in this model, a greater R_(L) has been observed followingthe second challenge with a given allergen dose than the first; this isantigen priming. The two antigen challenges caused AHR, as measured byan increased area under the histamine dose-response curve and/or a fallin PC₃₀, and BAL, as well as eosinophilia at day 11 compared to day 1.Animals displaying an AHR-phenotype were selected to enter phase II.

Phase II was run exactly as phase I except that all animals received a30 mg/kg BAK502G9 infusion on days 1, 5 and 9. The effects of BAK502G9were assessed by comparing the changes seen in phase II with changesseen in phase I for individual animals.

Blood, serum, BAL and lung tissue were colleted. Serum IgE levels weremonitored by ELISA. Serum from BAK502G9 treated cynomolgus monkeys wasshown to inhibit the expression of CD23 on human peripheral bloodmononuclear cells induced by IL-13 but not IL-4. The magnitude of thisinhibition was consistent with the serum BAK502G9 levels predicted by PKELISA.

Results

BAK502G9 significantly inhibited AHR as measured by R_(L) AUC (p<0.05)(FIG. 26A; Table 7). An inhibitory effect of BAK502G9 on AHR, asmeasured by PC₃₀, was observed but did not reach statisticalsignificance (FIG. 26B; Table 7). BAK502G9 also significantly inhibitedboth antigen priming (p<0.01) (FIG. 26C; Table 7) and BAL inflammation.BAK502G9 significantly inhibited total cell (p<0.05) and eosinophil(p<0.05) but not macrophage, lymphocyte or mast cell influx into the BAL(FIG. 26D; Table 7).

EXAMPLE 24

Efficacy of Anti-Human IL-13 Antibodies Against the Asthmatic Phenotypethat Develops When Human IL-13 is Administered to the Mouse Lung

Murine model of airways hyperresponsiveness

The efficacy of the anti-human IL-13 neutralising antibody BAK502G9,against the development of airways hyper-responsiveness (AHR) followingadministration of human IL-13 to the mouse lung was investigated. Thismodel was performed essentially as described by Yang et al [119] withthe exception that human IL-13 was used in place of murine IL-13.

Model Protocol

To develop the phenotype, male BALB/c mice were exposed to two doses ofhuman IL-13 separated by a 48-hour interval. In brief, mice wereanaesthetised with an intravenous injection of 100 μl saffan solution(1:4 diluted in water). Mice were intubated with a 22-gauge catheterneedle, through which human recombinant IL-13 (25 μg dissolved in 20 μlphosphate-buffered saline (PBS)) or vehicle control (PBS) was instilled.Airway function was assessed 24 hours after the last administration ofIL-13 by increasing methacholine challenges and monitored usingconscious plethysmography (Buxco). PC₂₀₀ (concentration of methacholinerequired to increase baseline penH by 200%) was determined from 4parameter unfixed curve fitting of methacholine dose-response curves.Antibody treatments were administered by intra-peritoneal injection 24hours prior to the each dose of IL-13.

Results

Intratracheal installation of human IL-13 into naive wild-type miceresulted in development of significant (p<0.05) airwayshyperresponsiveness relative to control animals as determined by PC₂₀₀methacholine concentrations. Systemically administered BAK502G9 (lmg/kg)significantly (p<0.01) inhibited the development of AHR whereas the nullcontrol antibody had no effect (FIG. 23).

EXAMPLE 25

Neutralisation Potency of BAK502G9 as a Human IgG4 Against Human IL-13Dependent IgE Release from Human B Cells.

B Cell Switching Assay Protocol

IL-13 has been shown to induce IgE synthesis in human B cells in vitro[120]. Factor dependent IgE release from human B cells was determined byELISA. The neutralisation potency of BAK502G9 as a human IgG4 wasassessed against human IL-13 dependent IgE release from human B cells.

Peripheral blood mononuclear cells (PBMC) were purified from human buffycoat (Blood Transfusion Service) by centrifugation over a 1.077 g/Ldensity gradient. B cells were purified from PBMC with a B cellisolation kit II (Miltenyi Biotec), using reagents and methods describedby the manufacturer. Assay media comprised Iscoves modified dulbeccosmedium (Life Technologies) containing 10% foetal bovine serum and 20μg/mL human transferrin (Serologicals Proteins Inc). Followingpurification, B cells were resuspended to a final concentration of10⁶/mL in assay media. 50 μl of resuspended cells were added to eachassay point in a 96 well assay plate. 50 μl of 4 μg/mL of the anti-CD40antibody EA5 (Biosource) was added to assay wells as appropriate. Testsolutions of antibodies (six replicates) were diluted to the desiredconcentration in assay media. An irrelevant antibody not directed atIL-13 was used as a negative control. 50 μl/well of the appropriate testantibody were added to the cells. Recombinant bacterially derived humanIL-13 (Peprotech) was subsequently added to a final concentration of 30ng/ml to give a total assay volume of 200 μl/well. The concentration ofIL-13 used in the assay was selected to give a maximal response. Assayplates were incubated for 14 days at 37° c under 5% CO₂. IgE levels inthe supernatant were determined by ELISA using reagents and protocolssupplied by the manufacturer (BD Biosciences, Bethyl Laboratories). Datawere analysed using Graphpad prism software.

Results

As demonstrated in FIG. 24, BAK502G9 (VH SEQ ID NO: 15; VL SEQ ID NO:16) was able to inhibit human IL-13 dependant IgE production by human Bcells. BAK502G9 as human IgG4 had an IC₅₀ of 1.8 nM against 30 ng/mlhuman IL-13.

EXAMPLE 26

Efficacy of BAK502G9 Against IL-13 Mediated Potentiation of HistamineInduced Ca²⁺ Signalling in Primary Human Bronchial Smooth Muscle Cells

IL-13 has been shown to directly modulate the contractility of airwaysmooth muscle [121, 122]. Intracellular calcium mobilization is aprerequisite for smooth muscle contraction. Recent studies have shownthat IL-13′s ability to alter smooth muscle contractility is mediated inpart through modulation of contractile agonist induced Ca²⁺ signaling[123, 124].

The efficacy of BAK502G9, an anti-human IL-13 antibody formatted as anIgG4, against IL-13 mediated alterations in primary human bronchialsmooth muscle cells (BSMC) signalling responses to the contractileagonist, histamine, was investigated in a Ca²⁺ signalling assay.

BSMC Ca²⁺ Signalling Assay Protocol

Human primary BSMC, Smooth Muscle Growth Medium-2 (SmGM-2) and SmoothMuscle Basal Medium (SmBM) were obtained from Bio Whittaker. The BSMCwere maintained in SmGM-2 according to supplier's recommendations. BSMCwere plated at 2×10⁴ cells/well in a 96-well microtitre cell cultureplate and were allowed to attach for 24 hours, then re-fed and incubatedfor a further 24 hours. Prior to the Ca²⁺ signalling experiment, theBSMC were stimulated with IL-13 (Peprotech) at 50 ng/ml finalconcentration with or without antibody and incubated for 18-24 hours.BAK502G9 and an isotype matched irrelevant control monoclonal antibody,CAT-001, were evaluated at a final concentration of 10 μg/ml. Changes inintracellular Ca²⁺ concentrations in response to histamine (Calbiochem),titrated from 20 μM, were measured using standard techniques with theCa²⁺ sensitive dye Fluo-4 (Molecular Probes) and a 96-well FluorescenceImaging Plate Reader (FLIPR) (Molecular Devices). The area under thecurve (AUC) of the Ca²⁺ signalling response to histamine was determinedfor each cell pre-treatment condition. Data analyses were performedusing GraphPad Prism version 4 for Windows (GraphPad Software).

Results

Pre-incubation of BSMC with IL-13 significantly enhanced Ca²⁺ signallingin response to histamine. Pre-incubation of BAK502G9 (FIG. 25B) (but notan irrelevant isotype control antibody (FIG. 25A)) with IL-13significantly inhibited the potentiation of Ca²⁺ signalling in responseto histamine (FIG. 25).

EXAMPLE 27

Neutralisation Potency of Anti-IL-13 Antibodies in a Human IL-13Dependent PBMC CD23 Expression Assay

The potency of a representative IL-13 antibody was evaluated in thehuman IL-13 dependent peripheral blood mononuclear cell (PBMC) CD23expression assay. PBMC respond to both IL-13 and IL-4 by increasing cellsurface expression of CD23 [120]. CD23 (FceRII) is the low-affinityreceptor for IgE and is expressed on a variety of inflammatory cells,including monocytes. Inhibition of human IL-13 dependent CD23 expressionupregulation was determined by measuring the reduction in binding offluorescently labelled CD23 monoclonal antibody to PBMCs by flowcytometry.

Assay Protocol

Human blood was obtained from the Blood Transfusion Service anderythrocytes depleted by 40 minute dextran-T500 (Pharmacia)sedimentation (0.6% final concentration). The leukocyte and plateletrich fraction was then separated by a 20 minute 1137 g centrifugationover a discontinuous Percoll gradient of 3 mL 64% and 5 mL 80% (100% was9 parts Percoll and 1 part 10×PBS). PBMCs were harvested from the top ofthe 64% layer, washed and resuspended in assay buffer (Invitrogen RPMI1640, 10% FCS, 200 IU/mL penicillin, 100 μg/mL streptomycin, 2 nML-Glutamine). The assay was performed in 24 well plates with 2×10⁶cells, ±80 μM recombinant human IL-13 (Peprotech) or 21 μM recombinanthuman IL-4 (R&D Systems), ±BAK502G9 or irrelevant IgG4, in a finalvolume of 500 mcL. Cells were cultured for 48 h at 37 C before beingharvested and stained with CD23-PE (BD Pharmingen) for 20 minutes at 4C. Finally, cells were read on a flow cytometer. CD23 expression wasdetermined by CD23 ‘score’; percent of CD23 positive cells multiplied bythe ‘brightness’ of the stain (geomean fluorescence). No stimulant CD23‘score’ was subtracted and data presented as a percentage of theresponse to IL-13 alone (100%). Data has been expressed as the mean±SEMdrawn from 4-6 separate experiments, using cells from 4-6 individualdonors, performed in triplicate for each point.

Results

Incubation of PBMC with 80 pM IL-13 or 21 pM IL-4 for 48 hours resultedin clear CD23 expression (FIG. 27 and FIG. 28). BAK502G9dose-dependently inhibited IL-13-induced CD23 expression with ageometric mean of 120.2 pM (FIG. 27). In contrast, BAK502G9 was unableto inhibit the CD23 expression induced by 21.4 pM IL-4 (n=4 fromindividual donors, FIG. 28). Irrelevant IgG4 did not inhibit eitherIL-13 or IL-4 dependent CD23 expression on PBMC (FIG. 27 and FIG. 28).Co-stimulation of PBMC with 80 pM IL-13 and 21.4 pM IL-4, produced anadditive CD23 response. BAK502G9, but not CAT-001, reduced CD23expression levels to those seen with IL-4 stimulation alone (FIG. 28).

EXAMPLE 28

Neutralisation Potency of a Human IL-13 Antibody in a Human IL-13Dependent Eosinophil Shape Change Assay

The aims of this study were to evaluate the effect of IL-13 antibodieson eosinophil shape change induced by mediators released from NHLFfollowing stimulation with factors found in the lungs of asthmatics suchas IL-13 [125,126], TNF-α [127], TGF-β1 [128]. IL-13 synergises withTNF-α [129] or TGF-β1 [130] to induce fibroblasts to produce eotaxin-1,which can then act to directly chemoattract eosinophils. Leukocyte shapechange responses are mediated through rearrangements of the cellularcytoskeleton and are essential to the processes of leukocyte migrationfrom the microcirculation into sites of inflammation. Inhibition ofIL-13-dependent shape-change-inducing factor release by NHLFs wasdetermined by measuring the reduction in eotaxin-lsecretion by ELISA andreduction in eosinophil shape change by flow cytometry.

Assay Protocol

NHLF cells were cocultured with media alone or media containingstimulants (9.6 nM IL-13, 285.7 pM TNF-α (R&D Systems) and 160 pM TGF-β1(R&D Systems) in the absence or presence of BAK502G9 (concentrationrange 875 nM-6.84 nM). Cells were then cultured for a further 48 h at37° C. before the resulting conditioned media was aspirated and storedat −80° C. The concentration of eotaxin-1 in conditioned media wasassessed using the R&D systems Duoset ELISA system (R&D Systems).

Human blood was obtained from the Blood Transfusion Service anderythrocytes depleted by 40 minute dextran-T500 (Pharmacia)sedimentation (0.6% final concentration). The leukocyte and plateletrich fraction was then separated by a 20 minute 1137 g centrifugationover a discontinuous Percoll gradient of 3 mL 64% and 5 mL 80% (100% was9 parts Percoll and 1 part 10×PBS). Granulocytes were harvested from the64%:80% interface, washed and resuspended in assay buffer (Sigma PBS, 1mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM D-glucose, 0.1% Sigma BSA, pH7.3). The assay was performed in FACS tubes with 5×10⁵ cells, ±3 nMrecombinant human eotaxin-1 (R&D Systems) or conditioned media, in afinal volume of 400 μL. Cells were incubated for 8.5 minutes at 37 Cbefore being transferred to 4° C. and fixed with a fixing agent(CellFix, BD Biosciences) and finally read on a flow cytometer.Eosinophils were identified by their FL-2 autofluorescence and theforward scatter (FSC) parameter read. Eosinophil FSC changed in responseto both eotaxin-1 and conditioned media providing measurement of shapechange. Tubes were sampled at high flow rate and acquisition wasterminated after 1000 eosinophil events or 1 minute, whichever was thesooner. Shape change was calculated as a percentage of the FSC caused byshape change buffer alone (100% blank shape change). Data have beenexpressed as the mean % blank shape change±SEM drawn from 4 separateexperiments. Each experiment used cells from an individual buffy coat(and hence individual donor), performed in duplicate for each point.

Results

NHLF cells co-stimulated with 9.6 nM IL-13, 285.7 pM TNF-α and 160 pMTGF-β1 and cultured for 48 h secreted 9.6 nM eotaxin-1 into the culturemedia. In contrast, NHLF cells cultured only with maintenance mediasecreted 0.1 nM eotaxin-1 into the culture media. This eotaxin-1production was IL-13 dependent as IL-13/TNF-α/TGF-β1 co-stimulated NHLFcell eotaxin-1 production was dose-dependently inhibited by BAK502G9with an IC₅₀ of 32.4 nM (FIG. 29A).

The primary aim of this part of the study was to examine eosinophilshape change. The magnitude of eosinophil shape change in response to 3nM eotaxin (positive control) was 122.2±2.1% (n=4). Eotaxin-1 inducedshape change was completely inhibited by 100 nM of an anti-eotaxinantibody CAT-213, mean shape change 101.0±1.0% (n=4).

Media from NHLF cells co-stimulated with 9.6 nM IL-13, 285.7 pM TNF-αand 160 pM TGF-β1 and cultured for 48 h (conditioned media), induced aclear eosinophil and shape change (FIG. 29B). In contrast, media fromNHLF cultured for 48 h in NHLF maintenance media alone did not induceeosinophil shape change (FIG. 29B).

The addition of anti-IL-13 antibody BAK502G9 to co-stimulated mediaprior to NHLF culture, resulted in a dose-dependent inhibition ofeosinophil shape change, with a geometric mean IC₅₀ of 16.8 nM whenassayed at 1:16 dilution (FIG. 29B).

The ability of stimulants (IL-13, TNF-α and TGF-β1) not cultured withNHLF cells to induce eosinophil and neutrophil shape change was alsoinvestigated. 9.6 nM IL-13, 285.7 pM TNF-α and 160 pM TGF-β1 did notinduce a clear eosinophil shape change. This suggests that theeosinophil shape change ability of conditioned media develops duringNHLF cell culture with the stimulants is not due to any of thestimulants alone or in combination (FIG. 29B).

Example 29

Mapping of Anti-IL-13 Antibodies on Human IL-13

The epitope mapping of a representative IL-13 antibody BAK502G9 wasperformed using a molecular approach and standard peptide excision.

Molecular Approach

IL-13 chimaeras were engineered, where parts of the human IL-13 sequencewere replaced with murine sequence. These chimeras were used in bindingstudies with representative IL-13 antibodies to help identify thespecific epitope.

Two panels of IL-13 chimaeras were produced. The first panel containednine chimaeras (FIG. 30) and was used to locate the general position ofthe epitope. The second panel contained ten chimaeras (FIG. 31) and wasused to fine map the epitope.

The chimaeric IL-13 sequences were assembled using PCR and cloned into aGateway® entry vector, which were then recombined with a destinationvector pDEST8 (modified to code for a detection and affinity tag at theC-terminus of the recombinant protein). These expression vectors wereused to transform DH10Bac™ chemically competent E coli allowingsite-specific transposition of tagged chimeric IL-13, into thebaculovirus shuttle vector (bacmid). Recombinant bacmid DNA was isolatedfor each IL-13 chimera and transfected into Sf9 (Spodoptera frugiperda)insect cells using Cellfectin® Reagent. Recombinant baculovirus washarvested 72 hours post-transfection and passaged through Sf9 insectcells twice more.

Insect 2000-500 ml culture supernatant was purified on an affinitycolumn and eluted material was concentrated from 16 to 1 ml and loadedon a size exclusion Superde×200 HR10/300GL column for final polishingand buffer exchange.

A homogenous competition assay using biotinylated human IL-13,streptavidin-anthophyocynate and Europium labelled BAK502G9 wasdeveloped. The assay is as follows: Eu-BAK502G9 binds biotinylated-humanIL-13, the complex is then recognised by the streptavidin APC conjugateand when a flash of light is applied the energy is transferred from theAPC label to the Europium by proximity, and time resolved florescencecan be measured. Competition for this binding is introduced by way ofthe un-labelled human IL-13 (as control) and the chimeric constructs.This competition is quantified to calculate the relative affinities ofthe IL-13 mutants for IL-13 antibodies enabling mutations alteringbinding to be identified.

Results

Chimeric construct IL13-Helix D (Table 5) was found to be the weakestcompetitor against biotinylated human IL-13 for binding BAK502G9,indicating that helixD within the IL-13 molecule was involved withBAK502G9 epitope binding (Table 5) Reduced activity was also seen forthe 4041 and 3334 mutants where residues 40, 41, and 33, 34 of theparent sequence respectively were changed indicating potentialinvolvement of helixA in the recognition of BAK502G9. The reducedactivities of loop3 was discounted as this loop has a reduced number ofamino acids in the mutant as compared to the human molecule and islikely to alter the overall structure of the protein. Other reductionsin the ability of the chimeric IL-13 molecules to compete for BAK502G9binding were not considered significant for such amino acid changes.

A more targeted set of mutations within helix D (FIG. 26) were thentested. Results obtained are demonstrated in Table 6 and are as follows:

Results show that chimeric constructs 116117TK (where lysine at position116 was replaced with threonine and the aspartate at position 117 wasreplaced with lysine), 123KA (where lysine at position 123 was replaced)and 127RA (where arginine at position 127 was replaced) are least ableto compete for binding to BAK502G9 (123KA and 127RA do not compete at 1pM). Other residues implicated in binding to BAK502G9 due to theirreduced effectiveness in the competition assay include the helixDresidues 124Q (here lysine has been replaced with glutamine) and120121SY (a leucine histidine pair has been changed to a serine tyrosinepair). Mutation of leucine at position 58 L also reduces binding andanalysis of the 3D structures revealed that this residue packs againsthelixD and may either be directly contacted by BAK502G9 or may affectthe alignment of helixD.

These experiments demonstrate that residues within helixD are criticalfor the binding of BAK502G9 to IL-13. In particular the lysine atposition 123 and the arginine at position 127 are critical for thisbinding as mutation to either abolishes binding of BAK502G9.

Epitope Excision

The epitope mapping of BAK502G9 was also performed using the standardpeptide excision procedure. Here IgG is immobilised onto solid phase andallowed to capture the IL-13 ligand. The formed complex is then subjectto specific proteolytic digestion, during which accessible peptide bondsare cleaved, however those protected by the IgG: ligand interface remainintact. Thus, a peptide containing the epitope remains bound to the IgG.This can then be desorbed, collected and identified by mass spectrometry(ms).

Two complementary techniques were used, the first made use of theCiphergen ProteinChip Reader MALDI-TOF mass spectrometer, where it waspossible to covalently link the IgG to a mass spectrometer chip and thenperform the digestion and extraction in-situ. The second technique usedbiotinylated BAK502G9 linked to streptavidin coated beads and allowedthe collection of sufficient peptide for sequence confirmation by tandemmass spectrometry (ms/ms).

The two procedures although differing in absolute detail and scaleinvolved essentially the same steps, coupling of the IgG, blocking ofunreacted binding sites, washing, ligand capture, removal of unboundligand, digestion and a final washing step.

The MALDI-TOF ms approach made use of proprietary ms chips activatedwith carbonyldiimidazole that covalently binds to free primary aminegroups to which the IgG at 1-2 mg/ml in PBS was coupled to overnight at4° C. The chip was subsequently blocked with an ethanolamine solution atroom temperature for 1 hour and then washed extensively with PBS or HBSplus a suitable detergent. A one picomole aliquot of IL-13 was thenapplied to the chip in either PBS or HBS and allowed to bind to thechemically immobilized IgG for 2 hours at room temperature. This wasfollowed by further washes in PBS or HBS with and without detergent toremove any non-specifically bound IL-13. A solution of trypsin rangingfrom 200 to 3.1 μg/ml in PBS or HBS was then applied to the IgG:ligandcomplex and digestion allowed to proceed for 30 minutes at roomtemperature after which the chip was washed in PBS or HBS plusdetergent, PBS or HBS and finally water. After application of a suitableMALDI-TOF ms matrix the chip was then be placed directly in the massspectrometer and analysed.

The bead based approach started with the biotinylation of the IgG, usingan NHS biotin compound, at a molar ratio of 1 IgG to 4 biotin molecules.Removal of unbound biotin and the by-products of the reaction using gelfiltration followed this. The biotinylated IgG was then allowed to bindto neutravidin coated agarose beads, where it was attempted to maximizethe IgG capture. Aliquots of IgG coated beads were then dispensed into aconcentrator spin columns and washed with Dulbecco's PBS+0.05% Tween 20followed by resuspension in Dulbecco's PBS+0.05% Tween 20. A pulse ofIL-13 was then applied to the resuspended IgG beads and binding allowedto proceed for 10 minutes after which the liquid phase was removed bycentrifugation and the beads washed with Dulbecco's PBS+0.05% Tween 20followed by resuspension in Dulbecco's PBS+0.05% Tween 20.

The bead:IgG:ligand complex was then subject to proteolysis with eithertrypsin or chymotrypsin with incubation at room temperature or 37° C.After which the beads were again washed in Dulbecco's PBS+0.05% Tween 20followed by a further washes in Dulbecco's PBS without detergent. Thebeads were then resuspended in a water, acetonitrile, trifluroacetic mixand the supernatant recovered. This was then variously analysed eitherby MALDI-TOF ms or by reverse phase HPLC mass spectrometry, includingtandem (ms/ms) fragmentation using the ThermoQuest LCQ ESI ion-trap massspectrometer. An attempt was then made to match the resultingfragmentation pattern to the human IL-13 sequence and the separate heavyand light chain sequence of BAK502G9 IgG.

During the experimental sequence a number of controls, primarily blanksurfaces, IgG only and isotype controls were employed to demonstratethat the identified peptides were derived specifically from IgG capturedIL-13 and not a product of BAK502G9 or non-specifically bound IL-13digestion.

Results

The experimental series consistently gave single IL-13 specific peptidesfor each digestion. Data from the LCQ ion trap instrument revealed thatthe tryptic fragment had a monoisotopic mass of 3258Da (MH+) and thechymotrypsin fragment a monoisotopic mass of 3937Da (MH+).

A search of these masses against the appropriate in silico digestion ofhuman IL-13 gave close matches to related peptides in the C-terminalportion of the molecule.

Match for Trypsin Peptide Mass: 3258Da

At a tolerance of 1000 ppm, 3258Da matches to the sequence from asparticacid at position 106 to the C-terminal asparagine at position 132. Thereare no other matches at this tolerance. This region is highlighted inbold on the sequence of the precursor form of human IL-13 below.

MALLLTTVIALTCLGGFASPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFN

Match for Chymotrypsin Peptide Mass: 3937Da

At a tolerance of 1000 ppm, 3937Da matches to the sequence from serineat position 99 to the C-terminal asparagine at position 132. This regionis highlighted in bold on the sequence of the precursor form of humanIL-13 below.

MALLLTTVIALTCLGGFASPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFN

Both these matches show that the BAK502G9 IgG retains the C-terminalportion of the IL-13 molecule during proteolysis of the antibody:ligandcomplex.

The identity of both peptides was successfully confirmed by the ms/ms,neither of which showed any significant sequence parallels withBAK502G9. The ms/ms fragment map tailored to identify either Y or B ionsmatched 26 of 104 possible ions in one charge state for the trypsinpeptide and 19 of 128 possible ions for the chymotrypsin peptide. Areview of all charge states shows identification of 23 of the 27 aminoacid residues for the trypsin fragment and 29 of the 33 residues for thechymotrypsin fragment. This is sufficient to confirm identity.

The experimental sequence as a whole has identified that part of theBAK502G9 epitope on human IL-13 as lying within the twenty-sevenC-terminal amino acid residues. These findings corroborate the findingof the molecular approach detailed above.

REFERENCES

-   1. McKenzie, A. N., et al. J Immunol, 1993. 150(12): p. 5436-44.-   2. Minty, A., et al. Nature, 1993. 362(6417): p. 248-50.-   3. Nakamura, Y., et al. Am J Respir Cell Mol Biol, 1996. 15 (5): p.    680-7.-   4. Robinson, D. S., et al. N Engl J Med, 1992. 326(5): p. 298-304.-   5. Walker, C., et al. Am J Respir Crit Care Med, 1994. 150(4): p.    1038-48.-   6. Humbert, M., et al. Am J Respir Crit Care Med, 1996. 154(5): p.    1497-504.-   7. Corrigan, C. J. and A. B. Kay Int Arch Allergy Appl    Immunol, 1991. 94(1-4): p. 270-1.-   8. Bentley, A. M., et al. Am J Respir Cell Mol Biol, 1993. 8(1): p.    35-42.-   9. Murata, T., et al. Int J Hematol, 1999. 69(1): p. 13-20.-   10. Andrews, A. L., et al. J Biol Chem, 2002. 277(48): p. 46073-8.-   11. Miloux, B., et al. FEBS Lett, 1997. 401(2-3): p. 163-6.-   12. Hilton, D. J., et al. Proc Natl Acad Sci U S A, 1996. 93(1): p.    497-501.-   13. Kuperman, D., et al. J Exp Med, 1998. 187(6): p. 939-48.-   14. Nelms, K., et al. Annu Rev Immunol, 1999. 17: p. 701-38.-   15. Zhang, J. G., et al. J Biol Chem, 1997. 272(14): p. 9474-80.-   16. Caput, D., et al. J Biol Chem, 1996. 271(28): p. 16921-6.-   17. Kawakami, K., et al. Blood, 2001. 97(9): p. 2673-9.-   18. Wood, N., et al. J Exp Med, 2003. 197(6): p. 703-709.-   19. Chiaramonte, M.G., et al. J Exp Med, 2003. 197(6): p. 687-701.-   20. Beasley, R., et al. J Allergy Clin Immunol, 2000. 105(2 Pt    2): p. S466-72.-   21. Peat, J. K. and J. Li J Allergy Clin Immunol, 1999. 103(1 Pt    1): p. 1-10.-   22. Society, B. T., British guideline on the management of    asthma.Thorax, 2003. 58 Suppl 1: p. i1-94.-   23. GINA, Global Strategy for Asthma Management and Prevention.    2002, National Insitute of Health.-   24. Milgrom, H., B. Bender, and F. Wamboldt. Ann Allergy Asthma    Immunol, 2002. 88(5): p. 429-31.-   25. Fish, L. and C. L. Lung, Adherence to asthma therapy. Ann    Allergy Asthma Immunol, 2001. 86(6 Suppl 1): p. 24-30.-   26. Bender, B. G. J Allergy Clin Immunol, 2002. 109(6 Suppl): p.    S554-9.-   27. Wills-Karp, M., et al. Science, 1998. 282(5397): p. 2258-61.-   28. Grunig, G., et al. Science, 1998. 282(5397): p. 2261-3.-   29. Venkayya, R., et al. Am J Respir Cell Mol Biol, 2002. 26(2): p.    202-8.-   30. Morse, B., et al. Am J Physiol Lung Cell Mol Physiol, 2002.    282(1): p. L44-9.-   31. Zhu, Z., et al. J Clin Invest, 1999. 103(6): p. 779-88.-   32. Walter, D. M., et al. J Immunol, 2001. 167(8): p. 4668-75.-   33. Cohn, L., J. S. Tepper, and K. Bottomly. J Immunol, 1998.    161(8): p. 3813-6.-   34. Taube, C., et al. J Immunol, 2002. 169(11): p. 6482-9.-   35. Yang, E. S., et al. J. Allergy Immunol., 2002. 109: p. A168.-   36. Blease, K., et al. J Immunol, 2001. 166(8): p. 5219-24.-   37. Heinzmann, A., et al. Hum Mol Genet, 2000. 9(4): p. 549-59.-   38. Howard, T. D., et al. Am J Hum Genet, 2002. 70(1): p. 230-6.-   39. Kauppi, P., et al. Genomics, 2001. 77(1-2): p. 35-42.-   40. Graves, P. E., et al. J Allergy Clin Immunol, 2000. 105(3): p.    506-13.-   41. Arima, K., et al. J Allergy Clin Immunol, 2002. 109(6): p.    980-7.-   42. van der Pouw Kraan, T. C., et al. Genes Immun, 1999. 1(1): p.    61-5.-   43. Humbert, M., et al. J Allergy Clin Immunol, 1997. 99(5): p.    657-65.-   44. Kotsimbos, T. C., P. Ernst, and Q. A. Hamid, Proc Assoc Am    Physicians, 1996. 108(5): p. 368-73.-   45. Komai-Koma, M., F. Y. Liew, and P. C. Wilkinson, J    Immunol, 1995. 155(3): p. 1110-6.-   46. Naseer, T., et al. Am J Respir Crit Care Med, 1997. 155(3): p.    845-51.-   47. Huang, S. K., et al. J Immunol, 1995. 155(5): p. 2688-94.-   48. Kroegel, C., et al. Eur Respir J, 1996. 9(5): p. 899-904.-   49. Ohshima, Y., et al. Pediatr Res, 2002. 51(2): p. 195-200.-   50. Hasegawa, M., et al. J Rheumatol, 1997. 24(2): p. 328-32.-   51. Hancock, A., et al. Am J Respir Cell Mol Biol, 1998. 18(1): p.    60-5.-   52. Lee, C. G., et al. J Exp Med, 2001. 194(6): p. 809-21.-   53. Jain-Vora, S., et al. Am J Respir Cell Mol Biol, 1997. 17(5): p.    541-51.-   54. Fallon, P. G., et al. J Immunol, 2000. 164(5): p. 2585-91.-   55. Chiaramonte, M. G., et al. J Clin Invest, 1999. 104(6): p.    777-85.-   56. Chiaramonte, M. G., et al. Hepatology, 2001. 34(2): p. 273-82.-   57. Sluiter, H. J., et al. Eur Respir J, 1991. 4(4): p. 479-89.-   58. Zheng, T., et al. J Clin Invest, 2000. 106(9): p. 1081-93.-   59. Tashkin, D. P., et al., Methacholine reactivity predicts changes    in lung function over time in smokers with early chronic obstructive    pulmonary disease. The Lung Health Study Research Group. Am J Respir    Crit Care Med, 1996. 153(6 Pt 1): p. 1802-11.-   60. Van Der Pouw Kraan, T. C., et al. Genes Immun, 2002. 3(7): p.    436-9.-   61. Skinnider, B. F., et al. Blood, 2001. 97(1): p. 250-5.-   62. Kapp, U., et al. J Exp Med, 1999. 189(12): p. 1939-46.-   63. Fiumara, P., F. Cabanillas, and A. Younes, Blood, 2001.    98(9): p. 2877-8.-   64. Terabe, M., et al. Nat Immunol, 2000. 1(6): p. 515-20.-   65. Ahlers, J. D., et al. Proc Natl Acad Sci U S A, 2002. 99(20): p.    13020-5.-   66. Hutchings, C., Generation of Naïve Human Antibody Libraries, in    Antibody Engineering, R. Kontermann and S. Dubel, Editors. 2001,    Springer Laboratory Manuals, Berlin. p. 93-108.-   67. Vaughan, T. J., et al. Nat Biotechnol, 1996. 14(3): p. 309-14.-   68. Kitamura, T., et al. Blood, 1989. 73(2): p. 375-80.-   69. Lefort, S., et al. FEBS Lett, 1995. 366(2-3): p. 122-6.-   70. Osbourn, J. K., et al. Immunotechnology, 1996. 2(3): p. 181-96.-   71. Howard, T. D., et al. Am J Respir Cell Mol Biol, 2001. 25(3): p.    377-84.-   72. Karlsson, R., A. Michaelsson, and L. Mattsson, J Immunol    Methods, 1991. 145(1-2): p. 229-40.-   73. Tomlinson, VBASE. 1997, MRC Centre for Protein Engineering,    Cambridge, UK.-   74. Altmann, F., et al. Glycoconj J, 1999. 16(2): p. 109-23.-   75. Drexler, H. G., et al. Leuk Res, 1986. 10(5): p. 487-500.-   76. Skinnider, B. F., U. Kapp, and T. W. Mak, Leuk Lymphoma, 2002.    43(6): p. 1203-10.-   77. Terada, N., et al. Clin Exp Allergy, 2000. 30(3): p. 348-55.-   78. Wenzel, S. E., et al. J Immunol, 2002. 169(8): p. 4613-9.-   79. Richter, A., et al. Am J Respir Cell Mol Biol, 2001. 25(3): p.    385-91.-   80. Bochner, B. S., et al. J Immunol, 1995. 154(2): p. 799-803.-   81. Kotowicz, K., et al. Int Immunol, 1996. 8(12): p. 1915-25.-   82. McKenzie, A. N., et al. Journal of Immunology, 1993. 150(12): p.    5436-44.-   83. Bouteiller, C. L., et al. J Immunol Methods, 1995. 181(1): p.    29-36.-   84. Riffo-Vasquez, Y., et al. Clin Exp Allergy, 2000. 30(5): p.    728-38.-   85. McMillan, S. J., et al. J Exp Med, 2002. 195(1): p. 51-7.-   86. Humbles, A. A., et al. Proc Natl Acad Sci U S A, 2002. 99(3): p.    1479-84.-   87. Temelkovski, J., et al. Thorax, 1998. 53(10): p. 849-56.-   88. Belvisi, M. G., et al., Pulm Pharmacol Ther, 2001. 14(3): p.    221-7.-   89. Barnes, P. J., et al. Eur Respir J, 1996. 9(4): p. 636-42.-   90. Barnes, P. J., Pharmacol Ther, 2003. 97(1): p. 87-94.-   91. Wardlaw, A. J., Clin Med, 2001. 1(3): p. 214-8.-   92. Edwards, J. C., et al. J Pathol, 1981. 134(2): p. 147-56.

93. McDonough, J. E., et al. W. M. Elliot, and J. C. Hogg. TGF-betaIsoform and IL-13 Immunostaining on Lung Tissue from Patients with COPD.in ATS 99th International Conference. 2003. Seattle.

-   94. Wold, et al. Multivariate data analysis in chemistry.    Chemometrics-Mathematics and Statistics in Chemistry (Ed.: B.    Kowalski), D. Reidel Publishing Company, Dordrecht, Holland, 1984    (ISBN 90-277-1846-6).-   95. Norman et al. Applied Regression Analysis. Wiley-Interscience;    3rd edition (April 1998) ISBN: 0471170828-   96. Abraham Kandel, Eric Backer. Computer-Assisted Reasoning in    Cluster Analysis. Prentice Hall PTR; (May 11, 1995), ISBN:    0133418847-   97. Wojtek Krzanowski. Principles of Multivariate Analysis: A User's    Perspective (Oxford Statistical Science Series, No 22 (Paper)).    Oxford University Press; (December 2000), ISBN: 0198507089-   98. Ian H. Witten, Eibe Frank. Data Mining: Practical Machine    Learning Tools and Techniques with Java Implementations. Morgan    Kaufmann; (October 11, 1999), ISBN: 1558605525-   99. David G. T. Denison (Editor), Christopher C. Holmes, Bani K.    Mallick, Adrian F. M. Smith. Bayesian Methods for Nonlinear    Classification and Regression (Wiley Series in Probability and    Statistics). John Wiley & Sons; (July 2002), ISBN: 0471490369-   100. Arup K. Ghose, Vellarkad N. Viswanadhan. Combinatorial Library    Design and Evaluation Principles, Software, Tools, and Applications    in Drug Discovery. ISBN: 0-8247-0487-8-   101. Chothia C. et al. Journal Molecular Biology (1992) 227,    799-817.-   102. Al-Lazikani, et al. Journal Molecular Biology (1997) 273(4),    927-948.-   103. Chothia, et al. Science, 233,755-758 (1986).-   104. Whitelegg, N. R. J. and Rees, A. R (2000). Prot. Eng., 13,    819-824.-   105. Available from Accelerys Inc.-   106. Guex, N. and Peitsch, M. C. (1997). Electrophoresis (1997) 18,    2714-2723.-   107. Kabat E A et al (1991): Sequences of Proteins of Immunological    Interest, 5^(th) Edition. US Department of Health and Human    Services, Public Service, NIH, Washington.-   108. Kontermann R and Dubel Stefan; (2001) Antibody Engineering,    Springer Laboratory Manuals.-   109. Mendez et al (1997); Nature Genetics Vol. 2: 146-156.-   110. Csonka E et al (2000) Journal of Cell Science, 113: 3207-3216.-   111. Vanderbyl S et al (2002) Molecular Therapy, 5(5): 10.-   112. Marasco WA (1997) Gene Therapy, 4(1): 11.-   113. Hanes J et al (2000). Methods in Enzymology, Vol 328:24.-   114. Li et al (2003). Abstract for poster [605] submitted at The    American Thoracis Society Annual Meeting, 2003, Seattle.-   115. Koide et al (1998). Journal of Molecular Biology, Vol    284:1141-1151.-   116. Nygren et al (1997). Current Opinion in Structural Biology, Vol    7:463-469.-   117. Heller, F., et al. (2002) Immunity, 17(5):629-38.-   118. Inoue, S., et al. (1999) Am J Gastroenterol, 94(9):2441-6.-   119. Yang, M., et al. Am J Respir Cell Mol Biol. 2001. 25(4): p.    522-30-   120. Punnonen J., et al 1993. Proc Natl Acad Sci. 90(8):3730-4.-   121.Grunstein, M., et al. Am J Physiol Lung Cell Mol Physiol 2002.    282: p. L520-L528.-   122. Laporte, J., et al. Am J Respir Crit Care Med 2001. 164: p.    141-148.-   123. Tliba O., et al. Br J Pharmacol 2003. 140(7): p. 1159-62.-   124. Deshpande, D., et al. Am J Respir Cell Mol Biol 2004. 31(1): p.    36-42; Epub Feb 5 as doi:10.1165/rcmb.2003-03130C.-   125. Humbert et al. 1997. J. Allergy Clin. Immunol., 99:657.-   126. Berry, M. A., Parker, D., Neale, N., Woodman, L., Morgan, A.    Monk, P. D. Submitted to J. Allergy Clin Immunol.-   127. Obase et al. Ann Allergy Asthma Immunol. 2001; 86(3):304-10.-   128. Chu et al. 2000; J. Allergy Clin. Immunol. 106:1115-   129. Terada et al. 2000. Clin. Exp. Allergy., 30: 348-55.-   130. Wenzel et al. 2000. J. Immunol. 169: 4613-19.

TABLE 1 Kabat HCDR1 HCDR2 numbering 31 32 33 34 35 50 51 52 52A 53 54 5556 57 58 59 60 61 62 63 64 65 BAK278D6 N Y G L S W I S A N N G D T N Y GQ E F Q G BAK502G9 BAK1187B4 PD D D D S T I BAK1167F2 PD Q T V BAK1105H3PD G L E L BAK1185E1 PD N D A T Q BAK1111D10 PD A T P D Q S BAK1184G5 PDR P T D L M BAK1166G2 PD T I BAK1184C8 PD G S Y S BAK1185F8 PD D R N I DY I BAK1167F4 PD D T V BAK1109G06 PD D D R T T Q BAK1183H4 PD N Y D G NQ BAK1183D2 PD R S D Q I BAK1184F9 PD G I D V L BAK1103G08 PD R A D E HBAK1157D08 PD L T V BAK1183B5 PD G N L BAK1097H06 PD G P S K E SBAK1106F04 PD R P R D T H BAK1183G5 PD G R S A L BAK1161H07 PD G T VBAK1162G04 PD E T I BAK1161D07 PD D T I BAK1162D09 PD G T I BAK1108F05PD E G S T N I BAK1107F08 PD F G P I M H BAK278D6 N Y G L S W I S A N NG D T N Y G Q E F Q G BAK502G9 ALA_VL26L O ALA_VL26M O ALA_VL26C OALA_VL26V O ALA_VL26K O ALA_VL26Y O ALA_VL26F O ALA_VL26R O ALA_VL26T OBAK1001C10 RD K BAK1018G7 RD D BAK1008C3 RD D BAK1009A4 RD G BAK1010D2RD BAK1007F9 RD BAK1010H9 RD R BAK1008D2 RD ALA_VL26A O BAK1007C4 RD GALA_VL26H O BAK1054C8 RD R BAK1050G7 RD D BAK1016F8 RD BAK1050D2 RDBAK1063D10 RD I BAK1060F6 RD T BAK1021D5 RD I R BAK278D6 N Y G L S W I SA N N G D T N Y G Q E F Q G BAK502G9 BAK1062E10 RD S ALA_VL26G OBAK1020C6 RD K BAK1022G9 RD G D BAK1063G4 RD I BAK0495G5 PD BAK1049B8 RDR BAK1006E5 RD BAK1063F1 RD G BAK0494B6 PD BAK1010C5 RD R BAK0496H4 PDBAK0501B6 PD BAK1049D7 RD BAK0531E2 PD BAK0433C4 PD BAK1008E9 RD TBAK0469C8 PD BAK0442E6 PD BAK1047E3 RD BAK0472D7 PD BAK1008A7 RDBAK1063H4 RD K BAK0495A4 PD BAK0782D5 PD BAK0502C3 PD BAK1007H6 RDBAK278D6 N Y G L S W I S A N N G D T N Y G Q E F Q G BAK502G9 BAK1004E6BAK1049G1 D BAK0464B2 BAK0502D5 Phage PD QDLGE T VIF DNA DGT DLAP STDTEQLY ILQSM R R KG R K Display RGE PNY TSIR GKI NVAMG HDK Ribosome RDDisplay Point O mutations Germlining GL Kabat HCDR3 LCDR1 numbering 9596 97 98 99 100 100A 100B 100C 100D 100E 101 102 linker 24 25 26 27 2829 30 31 32 33 34 BAK278D6 D S S S N W A R W F F D L G G N N I G S K L VH BAK502G9 S I BAK1187B4 PD S I BAK1167F2 PD S I BAK1105H3 PD S IBAK1185E1 PD S I BAK1111D10 PD N S I BAK1184G5 PD S I BAK1166G2 PD S IBAK1184C8 PD S I BAK1185F8 PD S I BAK1167F4 PD S I BAK1109G06 PD S IBAK1183H4 PD S I BAK1183D2 PD S I BAK1184F9 PD S I BAK1103G08 PD S IBAK1157D08 PD S I BAK1183B5 PD S I BAK1097H06 PD S I BAK1106F04 PD S IBAK1183G5 PD S I BAK1161H07 PD S I BAK1162G04 PD S I BAK1161D07 PD S IBAK1162D09 PD S I BAK1108F05 PD S I BAK278D6 D S S S N W A R W F F D L GG N N I G S K L V H BAK502G9 S I BAK1107F08 PD S I ALA_VL26L O S LALA_VL26M O S M ALA_VL26C O S C ALA_VL26V O S V ALA_VL26K O S KALA_VL26Y O S Y ALA_VL26F O S F ALA_VL26R O S R ALA_VL26T O S TBAK1001C10 RD I BAK1018G7 RD S G BAK1008C3 RD S R BAK1009A4 RD NBAK1010D2 RD S G BAK1007F9 RD D G BAK1010H9 RD S G BAK1008D2 RD N S GALA_VL26A O S A BAK1007C4 RD S S ALA_VL26H O S H BAK1054C8 RD GBAK1050G7 RD G BAK1016F8 RD S T BAK1050D2 RD N I BAK1063D10 RD GBAK278D6 D S S S N W A R W F F D L G G N N I G S K L V H BAK502G9 S IBAK1060F6 RD BAK1021D5 RD I BAK1062E10 RD S ALA_VL26G O S G BAK1020C6 RDS G BAK1022G9 RD N BAK1063G4 RD G BAK0495G5 PD S R BAK1049B8 RD S GBAK1006E5 RD T G BAK1063F1 RD S S BAK0494B6 PD D S G BAK1010C5 RD SBAK0496H4 PD Y BAK0501B6 PD D S G BAK1049D7 RD S S G BAK0531E2 PD IBAK0433C4 PD T A BAK1008E9 RD G BAK046908 PD N A BAK0442E6 PD NBAK1047E3 RD I G BAK0472D7 PD T R BAK1008A7 RD V G BAK1063H4 RD GBAK0495A4 PD D P R P BAK278D6 D S S S N W A R W F F D L G G N N I G S KL V H BAK502G9 S I BAK0782D5 PD R BAK0502C3 PD K G BAK1007H6 RD S GBAK1004E6 RD N Y BAK1049G1 RD S BAK0464B2 PD N S BAK0502D5 PD R D SPhage PD RD NDTP R SAI Y DS ILMCVKY V G R Display RPK FRTSAHG RibosomeRD Display Point O mutations Germlining GL Mean Kabat LCDR2 LCDR3 TF1TF1 numbering 50 51 52 53 54 55 56 89 90 91 92 93 94 95 95A 95B 96 97*(nM) reps BAK278D6 D D G D R P S Q V W D T G S D P V V 44 BAK502G9  8BAK1187B4 PD 0.1 2 BAK1167F2 PD 0.2 2 BAK1105H3 PD 0.2 3 BAK1185E1 PD0.2 2 BAK1111D10 PD 0.2 2 BAK1184G5 PD 0.2 2 BAK1166G2 PD 0.2 2BAK1184C8 PD 0.2 2 BAK1185F8 PD 0.2 2 BAK1167F4 PD 0.2 2 BAK1109G06 PD0.2 2 BAK1183H4 PD 0.2 2 BAK1183D2 PD 0.3 2 BAK1184F9 PD 0.3 2BAK1103G08 PD 0.3 3 BAK1157D08 PD 0.3 2 BAK1183B5 PD 0.3 2 BAK1097H06 PD0.4 2 BAK1106F04 PD 0.4 2 BAK1183G5 PD 0.5 2 BAK1161H07 PD 0.6 1BAK1162G04 PD 0.6 1 BAK1161D07 PD 0.6 1 BAK1162D09 PD 0.8 1 BAK1108F05PD 0.9 1 BAK278D6 D D G D R P S Q V W D T G S D P V V 44 BAK502G9  8BAK1107F08 PD 0.9 1 ALA_VL26L O 1.4 1 ALA_VL26M O 1.5 1 ALA_VL26C O 2.51 ALA_VL26V O 2.5 1 ALA_VL26K O 2.9 1 ALA_VL26Y O 3.1 1 ALA_VL26F O 3.51 ALA_VL26R O 3.8 1 ALA_VL26T O 3.9 1 BAK1001C10 RD 4.3 1 BAK1018G7 RD4.5 2 BAK1008C3 RD 5.2 2 BAK1009A4 RD 5.3 2 BAK1010D2 RD 5.4 2 BAK1007F9RD 6.4 1 BAK1010H9 RD 6.8 2 BAK1008D2 RD 6.9 1 ALA_VL26A O 7.2 1BAK1007C4 RD 7.6 2 ALA_VL26H O 7.7 1 BAK1054C8 RD 9.3 1 BAK1050G7 RD 9.52 BAK1016F8 RD N 10.0 1 BAK1050D2 RD 10.0 1 BAK278D6 D D G D R P S Q V WD T G S D P V V 44 BAK502G9  8 BAK1060F6 RD T 11.7 1 BAK1021D5 RD 12.0 1BAK1062E10 RD 14.4 1 ALA_VL26G O 14.4 1 BAK1020C6 RD I 15.0 1 BAK1022G9RD 17.0 1 BAK1063G4 RD 17.0 1 BAK0495G5 PD 17.0 1 BAK1049B8 RD 18.0 1BAK1006E5 RD 18.0 1 BAK1063F1 RD 20.0 1 BAK0494B6 PD 21.8 4 BAK1010C5 RD22.0 1 BAK0496H4 PD 22.0 3 BAK0501B6 PD 22.8 2 BAK1049D7 RD 23.0 1BAK0531E2 PD 23.2 3 BAK0433C4 PD 25.3 4 BAK1008E9 RD 27.0 1 BAK0469C8 PD28.2 5 BAK0442E6 PD 29.3 3 BAK1047E3 RD 30.0 1 BAK0472D7 PD 31.0 4BAK1008A7 RD 32.0 1 BAK1063H4 RD 34.0 1 BAK0495A4 PD 34.6 5 BAK278D6 D DG D R P S Q V W D T G S D P V V 44 BAK502G9  8 BAK0782D5 PD 39.0 1BAK0502C3 PD 39.3 3 BAK1007H6 RD 40.5 2 BAK1004E6 RD 45.0 2 BAK1049G1 RD46.0 2 BAK0464B2 PD 28.6 5 BAK0502D5 PD 29.3 4 Phage PD T N I DisplayRibosome RD Display Point O mutations Germlining GL

TABLE 2 Binding specificity of anti-human IL-13 antibodies Human IL-13Non-human Human IL-13 variant primate IL-13 BAK278D6 + + +BAK502G9 + + + BAK615E3 + − −

TABLE 3a Kinetic analysis of anti-human IL-13 antibodies Off-rateOn-rate KD IgG (s⁻¹) (M⁻¹ s⁻¹) (pM) BAK278D6 7.41e⁻³ 5.49e⁵ 13500BAK502G9 4.09e⁻⁴ 2.49e⁶ 178 BAK1167F2 4.05e⁻⁴ 2.99e⁶ 136 BAK1183H43.00e⁻⁴ 3.7e⁶ 81

TABLE 3b Kinetic analysis of anti-murine IL-13 antibodies Off-rateOn-rate KD IgG (s⁻¹) (M⁻¹ s⁻¹) (pM) BAK209B11 1.98e⁻² 3.9e⁶ 5100

TABLE 4 Pharmacokinetics of BAK502G9 in 4 allergic but non- challengedcynomolgus primates (2 male/2 female) after a single 10 mg/kg i.v bolusdose over 29 days. C_(max) 349.04 (t = 0.25 h) (μg/mL) Vd_(inf) 75.03<80 mL/kg, infers no (mL · kg⁻¹) tissue binding. Cl_(inf) 0.23 (mL ·hr⁻¹ · kg⁻¹) AUC_(inf) 42.99 (mg · h · mL⁻¹) AUC_(ext) 17.34 <30% soclearance and (%) vol. of distribution should be accurate. T_(0.5)223.55 (h) BAK502G9 levels in serum were measured by ELISA (mean data).Vd_(inf) = volume of distribution over time 0-infinity, calculated fromthe extrapolated AUC. Cl_(inf) = clearance over time 0-infinity,calculated from the extrapolated AUC. AUC_(inf) = area under the curve(measure of total drug exposure) over time 0-infinity, including anextrapolated term based on the elimination rate constant (k) and thelast observed serum drug concentration. AUC_(ext) = percentage of thetotal AUC that is extrapolated. T_(0.5) = Drug half-life in the terminalelimination phase.

TABLE 5 First set of Chimeric constructs Chimeric constructs IC50 nMBAK502G9 0.17 ± 0.07 loop1 0.71 ± 0.35 hum-flag 1.30 ± 0.18 30R 1.76 ±0.45 3738VN 1.89 ± 1.9  helixB 2.49 ± 0.88 helixC 4.11 ± 0.70 loop3 5.45± 3.96 4041 12.02 ± 1.3  3334 12.17 ± 1.2  helixD 110.07 ± 9.9  

TABLE 6 Second Set of Chimeric Constructs Chimeric Constructs IC50 nMBAK502G9 0.11 ± 0.04 113H 1.6 ± 0.5 128H 1.6 ± 1.0 119LA 1.96 ± 1.0 130P 2.22 ± 0.8  120121SY 4.73 ± 1.5  58LA 5.2 ± 2.0 124Q 18.7 ± 15.9116117T   82 ± 11.3 123KA none 127RA none

TABLE 7 Effects of BAK502G9 on various predefined endpoints. Phase IPhase II Parameter change N change N Endpoint AHR (R_(L) AUC) 0.020 ±0.003 14^(a) 0.004 ± 0.006 14^(a) −0.016 ± 0.006* AHR (PC₃₀) −1.343 ±0.318  18^(b) −1.061 ± 0.244  18^(b) 0.282 ± 0.179 Antigen priming 0.159± 0.033 20^(c) 0.033 ± 0.025 20^(c)  −0.126 ± 0.043** (R_(L) AUC) BALtotal cells 20.623 ± 3.160  21^(d) 14.597 ± 1.951  21^(d) −6.026 ±2.194* BAL eosinophils 18.453 ± 3.009  21^(d) 13.412 ± 1.737  21^(d)−5.041 ± 2.090* BAL mononuclear 2.050 ± 0.438 21^(d) 1.176 ± 0.48121^(d) −0.874 ± 0.506  cells 21 animals displaying AHR (PC₃₀) in Phase Iand an additional animal with an antigen priming phenotype were carriedforward for testing in Phase II (22 in total). Not every animal had AHRas measured by both AUC and PC₃₀. Only animals which displayed AHR inphase I and whose AHR was assessed in both Phase I and Phase II wereincluded in the AHR results. Statistical testing was performed usingInStat. Testing was a 2-way student's t-test against the null hypothesisthat the endpoint did include the number 0 (i.e. there was no change inphase II compared to phase I); *p < 0.05, **p < 0.01. Data are shown asarithmetic mean ± SEM (n = 14-21). ^(a)5 animals were excluded from theAUC analysis as they did not display AHR (increased AUC) in Phase I. 3further animals were excluded due to a technical failure in Phase IIairway function data collection. ^(b)3 animals were excluded from PC₃₀analysis due to a technical failure in Phase II airway function datacollection (same animals as in ^(a)). The additional animal with antigenpriming phenotype was excluded as it did not display PC₃₀ AHR in PhaseI. ^(c)2 animals were excluded from the antigen priming analysis asthere was a technical failure in Phase I airway function datacollection. ^(d)1 animal was excluded from the BAL analysis due tomarked BAL inflammation at study initiation.

BAK278D6 HEAVY CHAIN CDR1-SEQ ID NO 1: NYGLSCDR2-SEQ ID NO 2: WISANNGDTNYGQEFQG CDR3-SEQ ID NO 3: DSSSNWARWFFDLBAK278D6 LIGHT CHAIN CDR1-SEQ ID NO 4: GGNNIGSKLVHCDR2-SEQ ID NO 5: DDGDRPS CDR3-SEQ ID NO 6: QVWDTGSDPVV BAK502G9HEAVY CHAIN CDR1-SEQ ID NO 7: NYGLS CDR2-SEQ ID NO 8: WISANNGDTNYGQEFQGCDR3-SEQ ID NO 9: DSSSSWARWFFDL LIGHT CHAINCDR1-SEQ ID NO 10: GGNIIGSKLVH CDR2-SEQ ID NO 11: DDGDRPSCDR3-SEQ ID NO 12: QVWDTGSDPVV BAK278D6 HEAVY CHAIN DOMAIN SEQ ID NO 13:EVQLVQSGAEVKKPGASVKVSCKASGYTFRNYGLSWVRQAPGQGLEWMGWISANNGDINYGQEFQGRITMITETSINTAHMELRSLRSDDTAVYYCVRDSSSNWARWFFDLWGKGTMV TVSSBAK278D6 LIGHT CHAIN DOMAIN SEQ ID NO 14:SYVLIQPPSVSVAPGQTARIPCGGNNIGSKLVHWYQQKPGQAPVLVVYDDGDRPSGIPERFSGSNSGNTAILTISRIDAGDEADYYCQVWDIGSDPVVFGGGIKLIVL BAK502G9HEAVY CHAIN DOMAIN SEQ ID NO 15:QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGLSWVRQAPGQGLEWMGWISANNGDTNYGQEFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDSSSSWARWFFDLWGRGTLV TVSSBAK502G9 LIGHT CHAIN DOMAIN SEQ ID NO 16:SYVLTQPPSVSVAPGKTARITCGGNIIGSKLVHWYQQKPGQAPVLVIYDDGDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDTGSDPVVFGGGTKLTVL BAK278D6 HEAVY CHAINFR1-SEQ ID NO 17: EVQLVQSGAEVKKPGASVKVSCKASGYTFRFR2-SEQ ID NO 18: WVRQAPGQGLEWMGFR3-SEQ ID NO 19: RITMTTETSTNTAHMELRSLRSDDTAVYYCVR BAK278D6 LIGHT CHAINFR1-SEQ ID NO 20: SYVLTQPPSVSVAPGQTARIPCFR2-SEQ ID NO 21: WYQQKPGQAPVLVVYFR3-SEQ ID NO 22: GIPERFSGSNSGNTATLTISRIDAGDEADYYC BAK167A11HEAVY CHAIN DOMAIN SEQ ID NO 23:EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVGAAGEGYYGYWGRGILVTV S BAK167A11LIGHT CHAIN DOMAIN SEQ ID NO 24:NFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSAPTTVIYDDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSNNDVFGGGTKVTVL BAK209B11HEAVY CHAIN DOMAIN SEQ ID NO 25:QVQLQESGGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVSSISASGDSTFYADSVKGRFTISRDNNKNMVFLQVNSLRADDTAVYFCAKDWSQWLVGDAFDVWGRGIT VTVSSBAK209B11 LIGHT CHAIN DOMAIN SEQ ID NO 26:DIQLTQSPSTLSASVGDRVTITCRASQSVSLWVAWYQQRPGKAPKLLIYDGSTLQSGVPARFSGSGSGTEFTLTISSLQPDDFATYYCQQYKTFSTFGQGTKVEIKRA BAK502G9 HEAVY CHAINFR1-SEQ ID NO 27: QVQLVQSGAEVKKPGASVKVSCKASGYTFTFR2-SEQ ID NO 28: WYRQAPGQGLEWMGFR3-SEQ ID NO 29: RVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR BAK502G9 LIGHT CHAINFR1-SEQ ID NO 30: SYVLTQPPSVSVAPGKTARITCFR2-SEQ ID NO 31: WYQQKPGQAPVLVIYFR3-SEQ ID NO 32: GIPERFSGSNSGNTATLTISRVEAGDEADYYC BAK615E3HEAVY CHAIN DOMAIN SEQ ID NO 33:EVQLLESGGGLVQPGGSLRLSCAASGFIFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVGKATTEEGYYGYWGRGTLV TVSSBAK615E3 LIGHT CHAIN DOMAIN SEQ ID NO 34:NFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSAPTTVIYDDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSNNDVFGGGIKVIVL BAK1167F2HEAVY CHAIN DOMAIN SEQ ID NO 35:QVQLVQSGAEVKKPGASVKVSCKASGYTFEQTGVSWVRQAPGQGLEWMGWISANNGDTNYGQEFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDSSSSWARWFFDLWGRGTLV TVSSBAK1167F2 LIGHT CHAIN DOMAIN SEQ ID NO 36:SYVLTQPPSVSVAPGKTARITCGGNIIGSKLVHWYQQKPGQAPVLVIYDDGDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDTGSDPVVFGGGTKLTVL BAK1183H4HEAVY CHAIN DOMAIN SEQ ID NO 37:QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGLSWVRQAPGQGLEWMGWINYDGGNTQYGQEFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDSSSSWARWFFDLWGRGTLV TVSSBAK1183H4 LIGHT CHAIN DOMAIN SEQ ID NO 38:SYVLTQPPSVSVAPGKTARITCGGNIIGSKLVHWYQQKPGQAPVLVIYDDGDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDTGSDPVVFGGGTKLTVL BAK1105H3HEAVY CHAIN DOMAIN SEQ ID NO 39:QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGLSWVRQAPGQGLEWMGWISGLNGETLYGQEFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDSSSSWARWFFDLWGRGTLV TVSSBAK1105H3 LIGHT CHAIN DOMAIN SEQ ID NO 40:SYVLTQPPSVSVAPGKTARITCGGNIIGSKLVHWYQQKPGQAPVLVIYDDGDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDTGSDPVVFGGGTKLTVL BAK1111D10HEAVY CHAIN DOMAIN SEQ ID NO 41:QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGLSWVRQAPGQGLEWMGWIATPDGQTSYGQEFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDSNSSWARWFFDLWGRGTLV TVSSBAK1111D10 LIGHT CHAIN DOMAIN SEQ ID NO 42:SYVLTQPPSVSVAPGKTARITCGGNIIGSKLVHWYQQKPGQAPVLVIYDDGDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDTGSDPVVFGGGTKLTVL BAK1167F4HEAVY CHAIN DOMAIN SEQ ID NO 43:QVQLVQSGAEVKKPGASVKVSCKASGYTFIDTGVSWVRQAPGQGLEWMGWISANNGDTNYGQEFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDSSSSWARWFFDLWGRGTLV TVSSBAK1167F4 LIGHT CHAIN DOMAIN SEQ ID NO 44:SYVLTQPPSVSVAPGKTARITCGGNIIGSKLVHWYQQKPGQAPVLVIYDDGDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDTGSDPVVFGGGTKLTVL BAK1184C8HEAVY CHAIN DOMAIN SEQ ID NO 45:QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGLSWVRQAPGQGLEWMGWISGSNGYTSYGQEFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDSSSSWARWFFDLWGRGTLV TVSSBAK1184C8 LIGHT CHAIN DOMAIN SEQ ID NO 46:SYVLTQPPSVSVAPGKTARITCGGNIIGSKLVHWYQQKPGQAPVLVIYDDGDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDTGSDPVVFGGGTKLTVL BAK1185E1HEAVY CHAIN DOMAIN SEQ ID NO 47:QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGLSWVRQAPGQGLEWMGWINDATGDTQYGQEFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDSSSSWARWFFDLWGRGTLV TVSSBAK1185E1 LIGHT CHAIN DOMAIN SEQ ID NO 48:SYVLTQPPSVSVAPGKTARITCGGNIIGSKLVHWYQQKPGQAPVLVIYDDGDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDTGSDPVVFGGGTKLTVL BAK1185F8HEAVY CHAIN DOMAIN SEQ ID NO 49: QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYGLSWVRQAPGQGLEWMGWIRNIDGYTIYGQEFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDSSSSWARWFFDLWGRGTLV TVSSBAK1185F8 LIGHT CHAIN DOMAIN SEQ ID NO 50:SYVLTQPPSVSVAPGKTARITCGGNIIGSKLVHWYQQKPGQAPVLVIYDDGDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDTGSDPVVFGGGTKLTVL BAK1187B4HEAVY CHAIN DOMAIN SEQ ID NO 51:QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGLSWVRQAPGQGLEWMGWIDDDSGTTIYGQEFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDSSSSWARWFFDLWGRGTLV TVSSBAK1187B4 LIGHT CHAIN DOMAIN SEQ ID NO 52:SYVLTQPPSVSVAPGKTARITCGGNIIGSKLVHWYQQKPGQAPVLVIYDDGDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDTGSDPVVFGGGTKLTVL BAK1166G2HEAVY CHAIN DOMAIN SEQ ID NO 53:QVQLVQSGAEVKKPGASVKVSCKASGYTFANTGISWVRQAPGQGLEWMGWISANNGDTNYGQEFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDSSSSWARWFFDLWGRGTLV TVSSBAK1166G2 LIGHT CHAIN DOMAIN SEQ ID NO 54:SYVLTQPPSVSVAPGKTARITCGGNIIGSKLVHWYQQKPGQAPVLVIYDDGDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDTGSDPVVFGGGTKLTVL BAK167A11 HEAVY CHAINCDR1-SEQ ID NO 55: SYAMS CDR2-SEQ ID NO 56: AISGSGGSTYYADSVKGCDR3-SEQ ID NO 57: VGAAGEGYYGY BAK167A11 LIGHT CHAINCDR1-SEQ ID NO 58: TRSSGSIASNYVQ CDR2-SEQ ID NO 59: DDNQRPSCDR3-SEQ ID NO 60: QSYDSNNDV BAK1167F2 HEAVY CHAINCDR1-SEQ ID NO 61: QTGVS CDR2-SEQ ID NO 62: WISANNNGDTNYGQEFQGCDR3-SEQ ID NO 63: DSSSSWARWFFDL BAK1167F2 LIGHT CHAINCDR1-SEQ ID NO 64: GGNIIGSKLVH CDR2-SEQ ID NO 65: DDGDRPSCDR3-SEQ ID NO 66: QVWDTGSDPVV BAK1166G2 HEAVY CHAINCDR1-SEQ ID NO 67: NTGIS CDR2-SEQ ID NO 68: WISANNGDTNYGQEFQGCDR3-SEQ ID NO 69: DSSSSWARWFFDL BAK1166G2 LIGHT CHAINCDR1-SEQ ID NO 70: GGNIIGSKLVH CDR2-SEQ ID NO 71: DDGDRPSCDR3-SEQ ID NO 72: QVWDTGSDPVV BAK1184C8 HEAVY CHAINCDR1-SEQ ID NO 73: NYGLS CDR2-SEQ ID NO 74: WISGSNGYTSYGQEFQGCDR3-SEQ ID NO 75: DSSSSWARWFFDL BAK1184C8 LIGHT CHAINCDR1-SEQ ID NO 76: GGNIIGSKLVH CDR2-SEQ ID NO 77: DDGDRPSCDR3-SEQ ID NO 78: QVWDTGSDPVV BAK1185E1 HEAVY CHAINCDR1-SEQ ID NO 79: NYGLS CDR2-SEQ ID NO 80: WINDATGDTQYGQEFQGCDR3-SEQ ID NO 81: DSSSSWARWFFDL BAK1185E1 LIGHT CHAINCDR1-SEQ ID NO 82: GGNIIGSKLVH CDR2-SEQ ID NO 83: DDGDRPSCDR3-SEQ ID NO 84: QVWDTGSDPVV BAK1167F4 HEAVY CHAINCDR1-SEQ ID NO 85: DTGVS CDR2-SEQ ID NO 86: WISANNGDTNYGQEFQGCDR3-SEQ ID NO 87: DSSSSWARWFFDL BAK1167F4 LIGHT CHAINCDR1-SEQ ID NO 88: GGNIIGSKLVH CDR2-SEQ ID NO 89: DDGDRPSCDR3-SEQ ID NO 90: QVWDTGSDPVV BAK1111D10 HEAVY CHAINCDR1-SEQ ID NO 91: NYGLS CDR2-SEQ ID NO 92: WIATPDGQTSYGQEFQGCDR3-SEQ ID NO 93: DSNSSWARWFFDL BAK1111D10 LIGHT CHAINCDR1-SEQ ID NO 94: GGNIIGSKLVH CDR2-SEQ ID NO 95: DDGDRPSCDR3-SEQ ID NO 96: QVWDTGSDPVV BAK1183H4 HEAVY CHAINCDR1-SEQ ID NO 97: NYGLS CDR2-SEQ ID NO 98: WINYDGGNTQYGQEFQGCDR3-SEQ ID NO 99: DSSSSWARWFFDL BAK1183H4 LIGHT CHAINCDR1-SEQ ID NO 100: GGNIIGSKLVH CDR2-SEQ ID NO 101: DDGDRPSCDR3-SEQ ID NO 102: QVWDTGSDPVV BAK1185F8 HEAVY CHAINCDR1-SEQ ID NO 103: DYGLS CDR2-SEQ ID NO 104: WRINDGYTIYGQEFQGCDR3-SEQ ID NO 105: DSSSSWARWFFDL BAK1185F8 LIGHT CHAINCDR1-SEQ ID NO 106: GGNIIGSKLVH CDR2-SEQ ID NO 107: DDGDRPSCDR3-SEQ ID NO 108: QVWDTGSDPVV BAK278D6 HEAVY CHAIN-SEQ ID NO: 109CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACAAATTATGGTCTCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTAATAATGGCGACACAAATTATGGACAGGAATTCCAGGGCAGAGTCACCATGACCACAGATACATCCACGAGCACAGCCTACATGGAGTTGAGGAGCCTGAGATCTGACGACACGGCCGTTTATTACTGTGCGAGAGACTCCAGCAGCAACTGGGCCCGCTGGTTTTTCGATCTCTGGGGCCGGGGGACACTGGTC ACCGTCTCCTCABAK278D6 LIGHT CHAIN-SEQ ID NO: 110TCCTATGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGTAAGACGGCCAGGATTACCTGTGGGGGAAACAACATTGGAAGTAAACTTGTACACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCATCTATGATGATGGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAGGCCGGGGATGAGGCCGACTATTATTGTCAGGTGTGGGATACTGGTAGTGATCCCGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGT BAK502G9 HEAVY CHAIN-SEQ ID NO: 111CAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACAAATTATGGTCTCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTAATAATGGCGACACAAATTATGGACAGGAATTCCAGGGCAGAGTCACCATGACCACAGATACATCCACGAGCACAGCCTACATGGAGTTGAGGAGCCTGAGATCTGACGACACGGCCGTTTATTACTGTGCGAGAGACTCCAGCAGCAGCTGGGCCCGCTGGTTTTTCGATCTCTGGGGCCGGGGGACACTGGTC ACCGTCTCCTCABAK502G9 LIGHT CHAIN-SEQ ID NO: 112TCCTATGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGAAAGACGGCCAGGATTACCTGTGGGGGAAACATCATTGGAAGTAAACTTGTACACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCATCTATGATGATGGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAGGCCGGGGATGAGGCCGACTATTATTGTCAGGTGTGGGATACTGGTAGTGATCCCGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGT BAK1105H03 HEAVY CHAIN-SEQ ID NO: 113CAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACAAATTATGGTCTCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCTCCGGCTTGAACGGCGAGACATTGTATGGACAGGAATTCCAGGGCAGAGTCACCATGACCACAGATACATCCACGAGCACAGCCTACATGGAGTTGAGGAGCCTGAGATCTGACGACACGGCCGTTTATTACTGTGCGAGAGACTCCAGCAGCAGCTGGGCCCGCTGGTTTTTCGATCTCTGGGGCCGGGGGACACTGGTC ACCGTCTCCTCABAK1105H03 LIGHT CHAIN-SEQ ID NO: 114TCCTATGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGAAAGACGGCCAGGATTACCTGTGGGGGAAACATCATTGGAAGTAAACTTGTACACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCATCTATGATGATGGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAGGCCGGGGATGAGGCCGACTATTATTGTCAGGTGTGGGATACTGGTAGTGATCCCGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGT BAK1111D10 HEAVY CHAIN-SEQ ID NO: 115CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACAAATTATGGTCTCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCGCAACCCCAGACGGCCAGACAAGCTATGGACAGGAATTCCAGGGCAGAGTCACCATGACCACAGATACATCCACGAGCACAGCCTACATGGAGTTGAGGAGCCTGAGATCTGACGACACGGCCGTTTATTACTGTGCGAGAGACTCCAACAGCAGCTGGGCCCGCTGGTTTTTCGATCTCTGGGGCCGGGGGACACTGGTC ACCGTCTCCTCABAK1111D10 LIGHT CHAIN-SEQ ID NO: 116TCCTATGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGAAAGACGGCCAGGATTACCTGTGGGGGAAACATCATTGGAAGTAAACTTGTACACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCATCTATGATGATGGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAGGCCGGGGATGAGGCCGACTATTATTGTCAGGTGTGGGATACTGGTAGTGATCCCGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGT BAK1167F2 HEAVY CHAIN-SEQ ID NO: 117CAAGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTGAGCAGACCGGCGTCTCCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTAATAATGGCGACACAAATTATGGACAGGAATTCCAGGGCAGAGTCACCATGACCACAGATACATCCACGAGCACAGCCTACATGGAGTTGAGGAGCCTGAGATCTGACGACACGGCCGTTTATTACTGTGCGAGAGACTCCAGCAGCAGCTGGGCCCGCTGGTTTTTCGATCTCTGGGGCCGGGGGACACTGGTC ACCGTCTCCTCABAK1167F2 LIGHT CHAIN-SEQ ID NO: 118TCCTATGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGAAAGACGGCCAGGATTACCTGTGGGGGAAACATCATTGGAAGTAAACTTGTACACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCATCTATGATGATGGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAGGCCGGGGATGAGGCCGACTATTATTGTCAGGTGTGGGATACTGGTAGTGATCCCGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGT BAK1167F04 HEAVY CHAIN-SEQ ID NO: 119CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTATCGACACCGGGGTCTCCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTAATAATGGCGACACAAATTATGGACAGGAATTCCAGGGCAGAGTCACCATGACCACAGATACATCCACGAGCACAGCCTACATGGAGTTGAGGAGCCTGAGATCTGACGACACGGCCGTTTATTACTGTGCGAGAGACTCCAGCAGCAGCTGGGCCCGCTGGTTTTTCGATCTCTGGGGCCGGGGGACACTGGTC ACCGTCTCCTCABAK1167F04 LIGHT CHAIN-SEQ ID NO: 120TCCTATGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGAAAGACGGCCAGGATTACCTGTGGGGGAAACATCATTGGAAGTAAACTTGTACACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCATCTATGATGATGGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAGGCCGGGGATGAGGCCGACTATTATTGTCAGGTGTGGGATACTGGTAGTGATCCCGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGT BAK1183H4 HEAVY CHAIN-SEQ ID NO: 121CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACAAATTATGGTCTCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACTACGACGGCGGCAACACACAGTATGGACAGGAATTCCAGGGCAGAGTCACCATGACCACAGATACATCCACGAGCACAGCCTACATGGAGTTGAGGAGCCTGAGATCTGACGACACGGCCGTTTATTACTGTGCGAGAGACTCCAGCAGCAGCTGGGCCCGCTGGTTTTTCGATCTCTGGGGCCGGGGGACACTGGTC ACCGTCTCCTCABAK1183H4 LIGHT CHAIN-SEQ ID NO: 122TCCTATGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGAAAGACGGCCAGGATTACCTGTGGGGGAAACATCATTGGAAGTAAACTTGTACACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCATCTATGATGATGGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAGGCCGGGGATGAGGCCGACTATTATTGTCAGGTGTGGGATACTGGTAGTGATCCCGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGT BAK1184C8 HEAVY CHAIN-SEQ ID NO: 123CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACAAATTATGGTCTCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGGGAGCAACGGCTACACATCTTATGGACAGGAATTCCAGGGCAGAGTCACCATGACCACAGATACGTCCACGAGCACAGCCTACATGGAGTTGAGGAGCCTGAGATCTGACGACACGGCCGTTTATTACTGTGCGAGAGACTCCAGCAGCAGCTGGGCCCGCTGGTTTTTCGATCTCTGGGGCCGGGGGACACTGGTC ACCGTCTCCTCABAK1184C8 LIGHT CHAIN-SEQ ID NO: 124TCCTATGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGAAAGACGGCCAGGATTACCTGTGGGGGAAACATCATTGGAAGTAAACTTGTACACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCATCTATGATGATGGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAGGCCGGGGATGAGGCCGACTATTATTGTCAGGTGTGGGATACTGGTAGTGATCCCGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGT BAK1185E1 HEAVY CHAIN-SEQ ID NO: 125CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACAAATTATGGTCTCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACGACGCCACCGGCGACACACAGTATGGACAGGAATTCCAGGGCAGAGTCACCATGACCACAGATACATCCACGAGCACAGCCTACATGGAGTTGAGGAGCCTGAGATCTGACGACACGGCCGTTTATTACTGTGCGAGAGACTCCAGCAGCAGCTGGGCCCGCTGGTTTTTCGATCTCTGGGGCCGGGGGACACTGGTC ACCGTCTCCTCABAK1185E1 LIGHT CHAIN-SEQ ID NO: 126TCCTATGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGAAAGACGGCCAGGATTACCTGTGGGGGAAACATCATTGGAAGTAAACTTGTACACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCATCTATGATGATGGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAGGCCGGGGATGAGGCCGACTATTATTGTCAGGTGTGGGATACTGGTAGTGATCCCGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGT BAK1185F8 HEAVY CHAIN-SEQ ID NO: 127CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACAGATTATGGTCTCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTAGAGTGGATGGGATGGATCCGCAACATCGACGGCTACACAATTTATGGACAGGAATTCCAGGGCAGAGTCACCATGACCACAGATACATCCACGAGCACAGCCTACATGGAGTTGAGGAGCCTGAGATCTGACGACACGGCCGTTTATTACTGTGCGAGAGACTCCAGCAGCAGCTGGGCCCGCTGGTTTTTCGATCTCTGGGGCCGGGGGACACTGGTC ACCGTCTCCTCABAK1185F8 LIGHT CHAIN-SEQ ID NO: 128TCCTATGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGAAAGACGGCCAGGATTACCTGTGGGGGAAACATCATTGGAAGTAAACTTGTACACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCATCTATGATGATGGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAGGCCGGGGATGAGGCCGACTATTATTGTCAGGTGTGGGATACTGGTAGTGATCCCGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGT BAK1187B4 HEAVY CHAIN-SEQ ID NO: 129CAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACAAATTATGGTCTCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCGACGACGACAGCGGCACGACAATATATGGACAGGAATTCCAGGGCAGAGTCACCATGACCACAGATACATCCACGAGCACAGCCTACATGGAGTTGAGGAGCCTGAGATCTGACGACACGGCCGTTTATTACTGTGCGAGAGACTCCAGCAGCAGCTGGGCCCGCTGGTTTTTCGATCTCTGGGGCCGGGGGACACTGGTC ACCGTCTCCTCABAK1187B4  LIGHT CHAIN-SEQ ID NO: 130 TCCTATGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGAAAGACGGCCAGGATTACCTGTGGGGGAAACATCATTGGAAGTAAACTTGTACACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCATCTATGATGATGGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAGGCCGGGGATGAGGCCGACTATTATTGTCAGGTGTGGGATACTGGTAGTGATCCCGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGT BAK1166G02 HEAVY CHAIN-SEQ ID NO: 131CAAGTGCAGTTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTGCGAACACCGGGATCTCGTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTAATAATGGCGACACAAATTATGGACAGGAATTCCAGGGCAGAGTCACCATGACCACAGATACATCCACGAGCACAGCCTACATGGAGTTGAGGAGCCTGAGATCTGACGACACGGCCGTTTATTACTGTGCGAGAGACTCCAGCAGCAGCTGGGCCCGCTGGTTTTTCGATCTCTGGGGTCGGGGGACACTGGTC ACCGTCTCCTCABAK1166G02 LIGHT CHAIN-SEQ ID NO: 132TCCTATGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGAAAGACGGCCAGGATTACCTGTGGGGGAAACATCATTGGAAGTAAACTTGTACACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCATCTATGATGATGGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAGGCCGGGGATGAGGCCGACTATTATTGTCAGGTGTGGGATACTGGTAGTGATCCCGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGT BAK165E7 HEAVY CHAIN-SEQ ID NO: 133EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGLSWVRQAPGQGLEWMGWISANNGETNYGQEFQGRVIMITETPINTAHMELRSLTSDDTAVYYCVRDSSSNWARWYFDLWGQGTLV TVSSBAK165E7 LIGHT CHAIN-SEQ ID NO: 134SYVLIQPPSVSVAPGQTARIPCGGNNIGSKLVHWYQQKPGQAPVLVVYDDGDRPSGIPERFSGSNSGNTAILTISRIDAGDEADYYCQVWDIGSDPVVFGGGIKLIVLG BAK165E7 HEAVY CHAINCDR1-SEQ ID NO: 135 NYGLS CDR2-SEQ ID NO: 136 WISANNGETNYGQEFQGCDR3-SEQ ID NO: 137 DSSSNWARWYFDL BAK165E7 LIGHT CHAINCDR1-SEQ ID NO: 138 GGNNIGSKLVH CDR2-SEQ ID NO: 139 DDGDRPSCDR3-SEQ ID NO: 140 QVWDTGSDPVV BAK582F7 HEAVY CHAINCDR1-SEQ ID NO 141: SYAMS CDR2-SEQ ID NO 142: AISGSGGSTYYADSVKGCDR3-SEQ ID NO 143: VGAAGEGYYGY BAK582F7 LIGHT CHAINCDR1-SEQ ID NO 144: TRSSGSIASNYVE CDR2-SEQ ID NO 145: DDNQRPSCDR3-SEQ ID NO 146: QSYDSNNDV BAK612B5 HEAVY CHAINCDR1-SEQ ID NO 147: SYAMS CDR2-SEQ ID NO 148: AISGSGGSTYYADSVKGCDR3-SEQ ID NO 149: VGRATTDEGYYGY BAK612B5 LIGHT CHAINCDR1-SEQ ID NO 150: TRSSGSIASNYVQ CDR2-SEQ ID NO 151: DDNQRPSCDR3-SEQ ID NO 152: QSYDSNNDV BAK615E3 HEAVY CHAINCDR1-SEQ ID NO 153: SYAMS CDR2-SEQ ID NO 154: AISGSGGSTYYADSVKGCDR3-SEQ ID NO 155: VGKATTEEGYY BAK615E3 LIGHT CHAINCDR1-SEQ ID NO 156: TRSSGSIASNYVQ CDR2-SEQ ID NO 157: DDNQRPSCDR3-SEQ ID NO 158: QSYDSNNDV BAK0278D6 HEAVY CHAINCDR1-SEQ ID NO 159: AATTATGGTCTCAGCCDR2-SEQ ID NO 160: TGGATCAGCGCTAATAATGGCGACACAAATTAT GGACAGGAATTCCAGGGCCDR3-SEQ ID NO 161: GACTCCAGCAGCAACTGGGCCCGCTGGTTTTTC GATCTC BAK278D6LIGHT CHAIN CDR1-SEQ ID NO 162: GGGGGAAACAACATTGGAAGTAAACTTGTACACCDR2-SEQ ID NO 163: GATGATGGCGACCGGCCCTCACDR3-SEQ ID NO 164: CAGGTGTGGGATACTGGTAGTGATCCCGTGGTA BAK502G9HEAVY CHAIN CDR1-SEQ ID NO 165: AATTATGGTCTCAGCCDR2-SEQ ID NO 166: TGGATCAGCGCTAATAATGGCGACACAAATTATGGACA GGAATTCCAGGGCCDR3-SEQ ID NO 167: GACTCCAGCAGCAGCTGGGCCCGCTGGTTTTTCGATCTC BAK502G9LIGHT CHAIN CDR1-SEQ ID NO 168: GGGGGAAACATCATTGGAAGTAAACTTGTACACCDR2-SEQ ID NO 169: GATGATGGCGACCGGCCCTCACDR3-SEQ ID NO 170: CAGGTGTGGGATACTGGTAGTGATCCCGTGGTACH Domains-SEQ ID NO: 171ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVIVPSSSLGIKTYTCNVDHKPSNIKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDILMISRIPEVICVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK CL Domain-SEQ ID NO: 172QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

1-91. (canceled)
 92. An isolated nucleic acid molecule comprising apolynucleotide encoding an antibody or antigen-binding fragment thereofthat binds human interleukin-13 (IL-13), wherein the antibody orantigen-binding fragment thereof comprises a set of complementaritydetermining regions (CDRs), HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3,selected from the group consisting of: (i) the BAK1183H4 set of CDRs,defined wherein the HCDR1 has the amino acid sequence of NYGLS (SEQ IDNO: 97), the HCDR2 has the amino acid sequence of WINYDGGNTQYGQEFQG (SEQID NO: 98), the HCDR3 has the amino acid sequence of DSSSSWARWFFDL (SEQID NO: 99), the LCDR1 has the amino acid sequence of GGNIIGSKLVH (SEQ IDNO: 100), the LCDR2 has the amino acid sequence of DDGDRPS (SEQ ID NO:101), and the LCDR3 has the amino acid sequence of QVWDTGSDPVV (SEQ IDNO: 102); (ii) the BAK1167F02 set of CDRs, defined wherein the HCDR1 hasthe amino acid sequence QTGVS (SEQ ID NO: 61), the HCDR2 has the aminoacid sequence WISANNGDTNYGQEFQG (SEQ ID NO: 62), the HCDR3 has the aminoacid sequence DSSSSWARWFFDL (SEQ ID NO: 63), the LCDR1 has the aminoacid sequence GGNIIGSKLVH (SEQ ID NO: 64), the LCDR2 has the amino acidsequence DDGDRPS (SEQ ID NO: 65), and the LCDR3 has the amino acidsequence QVWDTGSDPVV (SEQ ID NO: 66); (iii) the BAK1111D10 set of CDRs,defined wherein the HCDR1 has the amino acid sequence NYGLS (SEQ ID NO:91), the HCDR2 has the amino acid sequence WIATPDGQTSYGQEFQG (SEQ ID NO:92), the HCDR3 has the amino acid sequence DSNSSWARWFFDL (SEQ ID NO:93), the LCDR1 has the amino acid sequence GGNIIGSKLVH (SEQ ID NO: 94),the LCDR2 has the amino acid sequence DDGDRPS (SEQ ID NO: 95), and theLCDR3 has the amino acid sequence of QVWDTGSDPVV (SEQ ID NO: 96); (iv)the BAK1166G02 set of CDRs, defined wherein the HCDR1 has the amino acidsequence NTGIS (SEQ ID NO: 67), the HCDR2 has the amino acid sequenceWISANNGDTNYGQEFQG (SEQ ID NO: 68), the HCDR3 has the amino acid sequenceDSSSSWARWFFDL (SEQ ID NO: 69), the LCDR1 has the amino acid sequenceGGNIIGSKLVH (SEQ ID NO: 70), the LCDR2 has the amino acid sequenceDDGDRPS (SEQ ID NO: 71), and the LCDR3 has the amino acid sequenceQVWDTGSDPVV (SEQ ID NO: 72); (v) the BAK1167F04 set of CDRs, definedwherein the HCDR1 has the amino acid sequence DTGVS (SEQ ID NO: 85), theHCDR2 has the amino acid sequence WISANNGDTNYGQEFQG (SEQ ID NO: 86), theHCDR3 has the amino acid sequence DSSSSWARWFFDL (SEQ ID NO: 87), theLCDR1 has the amino acid sequence GGNIIGSKLVH (SEQ ID NO: 88), the LCDR2has the amino acid sequence DDGDRPS (SEQ ID NO: 89), and the LCDR3 hasthe amino acid sequence QVWDTGSDPVV (SEQ ID NO: 90); (vi) the BAK1184C8set of CDRs, defined wherein the HCDR1 has the amino acid sequence NYGLS(SEQ ID NO: 73), the HCDR2 has the amino acid sequence WISGSNGYTSYGQEFQG(SEQ ID NO: 74), the HCDR3 has the amino acid sequence DSSSSWARWFFDL(SEQ ID NO: 75), the LCDR1 has the amino acid sequence GGNIIGSKLVH (SEQID NO: 76), the LCDR2 has the amino acid sequence DDGDRPS (SEQ ID NO:77), and the LCDR3 has the amino acid sequence QVWDTGSDPVV (SEQ ID NO:78); (vii) the BAK1185E1 set of CDRs, defined wherein the HCDR1 has theamino acid sequence NYGLS (SEQ ID NO: 79), the HCDR2 has the amino acidsequence WINDATGDTQYGQEFQG (SEQ ID NO: 80), the HCDR3 has the amino acidsequence DSSSSWARWFFDL (SEQ ID NO: 81), the LCDR1 has the amino acidsequence GGNIIGSKLVH (SEQ ID NO: 82), the LCDR2 has the amino acidsequence DDGDRPS (SEQ ID NO: 83), and the LCDR3 has the amino acidsequence QVWDTGSDPVV (SEQ ID NO: 84); and (viii) the BAK1185F8 set ofCDRs, defined wherein the HCDR1 has the amino acid sequence DYGLS (SEQID NO: 103), the HCDR2 has the amino acid sequence WRINDGYTIYGQEFQG (SEQID NO: 104), the HCDR3 has the amino acid sequence DSSSSWARWFFDL (SEQ IDNO: 105), the LCDR1 has the amino acid sequence GGNIIGSKLVH (SEQ ID NO:106), the LCDR2 has the amino acid sequence DDGDRPS (SEQ ID NO: 107),and the LCDR3 has the amino acid sequence QVWDTGSDPVV (SEQ ID NO: 108).93. The nucleic acid molecule according to claim 92, wherein the nucleicacid molecule is operably linked to a control sequence.
 94. A vectorcomprising the nucleic acid molecule according to claim
 93. 95. A hostcell transformed with the vector of claim
 94. 96. The host cell of claim95, wherein the host cell is a mammalian host cell.
 97. The mammalianhost cell of claim 96, wherein the host cell is a Chinese hamster ovary(CHO) cell, a HeLa cell, a baby hamster kidney cell, a NSO mousemelanoma cell, a YB2/0 rat myeloma cell, a human embryonic kidney cell,or a human embryonic retina cell.
 98. A method of making an antibody orantigen-binding fragment thereof that binds human interleukin-13 (IL-13)comprising culturing a host cell according to claim 95 under suitableconditions for producing the antibody or antigen-binding fragmentthereof.
 99. The method of claim 98, further comprising isolating theantibody or antigen-binding fragment thereof secreted from the hostcell.
 100. An isolated nucleic acid molecule comprising a polynucleotideencoding an antibody or antigen-binding fragment thereof that bindshuman interleukin-13 (IL-13), wherein the antibody or antigen-bindingfragment thereof comprises a set of heavy and light chain variableregions (VH and VL) from the group consisting of:(i) the BAK1183H4 VH sequence (SEQ ID NO: 37) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGLSWVRQAPGQGLEWMGWINYDGGNTQYGQEFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDSSSSWARWFF DLWGRGTLVTVSSand the BAK1183H4 VL sequence (SEQ ID NO: 38)SYVLTQPPSVSVAPGKTARITCGGNIIGSKLVHWYQQKPGQAPVLVIYDDGDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDTGSDPVVFGGGTKLTVL,(ii) the BAK1111D10 VH sequence (SEQ ID NO: 41)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGLSWVRQAPGQGLEWMGWIATPDGQTSYGQEFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDSNSSWARWFF DLWGRGTLVTVSSand the BAK1111D10 VL sequence (SEQ ID NO: 42)SYVLTQPPSVSVAPGKTARITCGGNIIGSKLVHWYQQKPGQAPVLVIYDDGDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDTGSDPVVFGGGTKLTVL,(iii) the BAK1167F02 VH sequence (SEQ ID NO: 35)QVQLVQSGAEVKKPGASVKVSCKASGYTFEQTGVSWVRQAPGQGLEWMGWISANNGDTNYGQEFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDSSSSWARWFF DLWGRGTLVTVSSand the BAK1167F02 VL sequence (SEQ ID NO: 36)SYVLTQPPSVSVAPGKTARITCGGNIIGSKLVHWYQQKPGQAPVLVIYDDGDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDTGSDPVVFGGGTKLTVL,(iv) the BAK1166G02 VH sequence (SEQ ID NO: 53)QVQLVQSGAEVKKPGASVKVSCKASGYTFANTGISWVRQAPGQGLEWMGWISANNGDTNYGQEFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDSSSSWARWFFD LWGRGTLVTVSS andthe BAK1166G02 VL sequence (SEQ ID NO: 54)SYVLTQPPSVSVAPGKTARITCGGNIIGSKLVHWYQQKPGQAPVLVIYDDGDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDTGSDPVVFGGGTKLTVL,(v) the BAK1167F04 VH sequence (SEQ ID NO: 43)QVQLVQSGAEVKKPGASVKVSCKASGYTFIDTGVSWVRQAPGQGLEWMGWISANNGDTNYGQEFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDSSSSWARWFFD LWGRGTLVTVSS andthe BAK1167F04 VL sequence (SEQ ID NO: 44)SYVLTQPPSVSVAPGKTARITCGGNIIGSKLVHWYQQKPGQAPVLVIYDDGDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDTGSDPVVFGGGTKLTVL,(vi) the BAK1184C8 VH sequence (SEQ ID NO: 45)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGLSWVRQAPGQGLEWMGWISGSNGYTSYGQEFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDSSSSWARWFFDL WGRGTLVTVSS andthe BAK1184C8 VL sequence (SEQ ID NO: 46)SYVLTQPPSVSVAPGKTARITCGGNIIGSKLVHWYQQKPGQAPVLVIYDDGDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDTGSDPVVFGGGTKLTVL,(vii) the BAK1185E1 VH sequence (SEQ ID NO: 47)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGLSWVRQAPGQGLEWMGWINDATGDTQYGQEFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDSSSSWARWFF DLWGRGTLVTVSSand the BAK1185E1 VL sequence (SEQ ID NO: 48)SYVLTQPPSVSVAPGKTARITCGGNIIGSKLVHWYQQKPGQAPVLVIYDDGDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDTGSDPVVFGGGTKLTVL, and(viii) the BAK1185F8 VH sequence (SEQ ID NO: 49)QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYGLSWVRQAPGQGLEWMGWIRNIDGYTIYGQEFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDSSSSWARWFFDL WGRGTLVTVSS andthe BAK1185F8 VL sequence (SEQ ID NO: 50)SYVLTQPPSVSVAPGKTARITCGGNIIGSKLVHWYQQKPGQAPVLVIYDDGDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDTGSDPVVFGGGTKLTVL.


101. The nucleic acid molecule according to claim 100, wherein thenucleic acid molecule is operably linked to a control sequence.
 102. Avector comprising the nucleic acid molecule according to claim
 101. 103.A host cell transformed with the vector of claim
 102. 104. The host cellof claim 103, wherein the host cell is a mammalian host cell.
 105. Themammalian host cell of claim 104, wherein the host cell is a Chinesehamster ovary (CHO) cell, a HeLa cell, a baby hamster kidney cell, a NSOmouse melanoma cell, a YB2/0 rat myeloma cell, a human embryonic kidneycell, or a human embryonic retina cell.
 106. A method of making anantibody or antigen-binding fragment thereof that binds humaninterleukin-13 (IL-13) comprising culturing a host cell according toclaim 103 under suitable conditions for producing the antibody orantigen-binding fragment thereof.
 107. The method of claim 106, furthercomprising isolating the antibody or antigen binding fragment thereofsecreted from the host cell.